A vaccine in combination with an immune checkpoint inhibitor for use in treating cancer

ABSTRACT

A polypeptide for use in medicine is provided. The polypeptide is administered simultaneously, separately or sequentially with an immune checkpoint inhibitor. The polypeptide comprises at least one polypeptide comprising a region of at least 12 amino acids of a self-antigen or a sequence having at least 80% identity to the region. The polypeptide is less than 100 amino acids in length.

FIELD OF THE INVENTION

The present invention relates to a polypeptide, a nucleic acid molecule,a T-cell receptor, or a T-cell displaying the T-cell receptor, and animmune checkpoint inhibitor for use in medicine. The invention alsorelates a method of treatment of cancer in a patient. The inventionfurther relates to a composition and a kit suitable for the treatment ofcancer.

BACKGROUND OF THE INVENTION

Cancer is a disease characterised by new and abnormal growth of cellswithin an individual. Cancer develops through a multi-step processinvolving several mutational events that allow cancer cells to develop,that is to say cells which display the properties of invasion andmetastasis.

Numerous approaches have been proposed for the treatment of cancer. Oneapproach is the use of antigenic peptides which comprise fragments oftumour associated antigens (i.e. peptide-based cancer vaccines). Suchantigenic peptides, when administered to an individual, elicit an MHCclass I or class II restricted T-cell response against cells expressingthe tumour associated antigens.

It is to be appreciated that in order for such T-cell responses tooccur, the antigenic polypeptide must be presented on an MHC molecule.There is a wide range of variability in MHC molecules in humanpopulations. In particular, different individuals have different HLAalleles which have varying binding affinity for polypeptides, dependingon the amino acid sequence of the polypeptides. Thus an individual whohas one particular HLA allele may have MHC molecules that will bind apolypeptide of a particular sequence whereas other individuals lackingthe HLA allele will have MHC molecules unable to bind and present thepolypeptide (or, at least, their MHC molecules will have a very lowaffinity for the polypeptide and so present it at a relatively lowlevel). Therefore, variability in MHC molecules in the human populationmeans that providing a peptide-based cancer vaccine with broadpopulation coverage is problematic because not all individuals willmount an immune response against a given antigen.

An alternative approach to the treatment of cancer is to target proteinsinvolved in immune checkpoints in order to modulate an individual'simmune response to cancer. Immune checkpoint mechanisms that normallydown-regulate the immune system in order to prevent excessive anduncontrolled immune responses include cytotoxic T-lymphocyte-associatedprotein 4 (CTLA-4) and programmed cell death protein 1 (PD-1). CLTA-4and PD-1 downregulate pathways of T-cell activation and in individualswith cancer, this can result in natural immune responses against cancersbeing down-regulated. Antibody-mediated blockade of these checkpoints isexpected to release the potency of the inhibited immune response andimprove survival rates. Blockade of CTLA-4 in a clinical setting, forinstance using the anti-CTLA-4 antibodies ipilimumab or tremelimumab,resulted in durable survival benefits in about 20% of patients withmetastatic melanoma (McDermott et al. Ann Oncol. 2013 24(10):2694-2698). Anti-CTLA-4 therapy has also been investigated in othercancers such as non-small cell lung cancer, pancreatic cancer, ovariancancer, lymphoma, gastric cancer and breast cancer (Postow et al., JClin Oncol. 2015 33(17):1974-82; Kyi & Postow, FEBS Lett. 2014588(2):368-76).

The best therapeutic peptide-based cancer vaccines are capable ofeliciting cancer specific immune responses in a majority of patients,typically 60-80% (Kyte et al. Clin Cancer Res. 2011 17(13):4568-80;Brunsvig et al. Cancer Immunol Immunother. 2006 55(12):1553-64).However, clinical responses as a result of peptide vaccination aretypically seen only in very few patients (Reviewed in Melero et al. NatRev Clin Oncol. 2014 11(9):509-24). It was therefore expected thatcombining peptide-based cancer vaccines with inhibition of immunecheckpoints would produce both enhanced immune responses against thevaccine and higher clinical response rates.

However, in a landmark Phase III clinical study combining ipilimumabwith a 9-mer gp100 melanoma peptide vaccine, the combination was nobetter than ipilimumab alone, and the vaccine alone had no protectiveeffect (Hodi et al. N Engl J Med. 2010 363(8):711-23). Very similarresults were observed with a combination of another melanoma vaccine,containing three 9-mer peptides derived from Mart 1, gp100 andTyrosinase, and ipilimumab (Sarnaik et al. Clin Cancer Res. 201117(4):896-906).

Again no additional clinical benefit was associated with thecombination, compared to the result with ipilimumab alone. T cellresponses to the individual peptide components of the vaccine were low(0-20%) and not associated with clinical responses. Together the trialsincluded 722 (647+75) patients and thus these data strongly indicatedthat the administration of an immune checkpoint inhibitor, in particulara CTLA-4 blockade, in combination with peptide-based cancer vaccinesdoes not increase immune responses or result in improved clinicalefficacy of such vaccines in humans. This is contrary to what wasexpected from experiments in mice (Williams et al. Clin Cancer Res.2013, 19(13) 3545-3555; Met et al. Cancer Lett. 2006 231(2):247-256).The present invention seeks to provide a solution to this problem.

WO2015/095811 relates to methods for the treatment of neoplasia, andmore particularly tumours, by administering to a subject a neoplasiavaccine comprising a plurality of neoplasia/tumour-specific neoantigensand at least one checkpoint inhibitor. It is to be appreciated thatWO2015/096811 specifically relates to personalised cancer vaccinescomprising tumour-specific neoantigens, which are created by thepersonal mutations found in each patient's tumour. The personalisedcancer vaccines disclosed in WO2015/095811 would not be suitable acrossa broad range of the population. Furthermore, no experimental data onthe combination of the personalised cancer vaccines and the checkpointinhibitors is provided in WO2015/095811 to support the efficacy of thiscombination in the treatment of cancer. This is significant, given thepreponderance of evidence indicating that the administration of animmune checkpoint inhibitor in combination with a peptide-based cancervaccine does not increase immune responses or result in improvedclinical efficacy in humans, as explained above.

WO2015/033140 relates to an immunogenic tumour antigen peptide-derivedcomposition and to the treatment of cancer using the composition. Theconcept of combining the composition with immunotherapies orimmunomodulators (for example, including agents to block immunecheckpoints) is disclosed in general terms. However, WO2015/033140 doesnot provide any experimental data on the combination of thepeptide-derived composition with immune checkpoint inhibitors. This issignificant, given the preponderance of evidence indicating that theadministration of an immune checkpoint inhibitor in combination with apeptide-based cancer vaccine does not increase immune responses orresult in improved clinical efficacy in humans, as explained above.Therefore, no enabling disclosure of the combination in the treatment ofcancer is provided in WO2015/033140.

WO2016/025647 relates to a method of treating cancer with a combinationof IL-2, a therapeutic antibody or fragment thereof, and a cancervaccine. Example 4 of WO2016/025647 relates to a quadruple combinationMSA-IL-2 plus anti-PD-1 antibody plus TA99 (an anti-Trp-1 antibody) plusa cancer vaccine (an amphiphile cancer vaccine targeting Trp-2) in aB16F10 melanoma mouse model. The cancer vaccine is noted on page 84 toelicit a CD8+ T cell response meaning that it was between 8 and 10 aminoacids in length. This length of peptide is equivalent to that used inthe cancer vaccines of Hodi et al. 2006 and Sarnaik et al. 2011 andwhich produced no additional clinical benefit in humans when combinedwith ipilimumab.

Yuan et al. Cancer Immunol Immunother. 2011 August; 60(8):1137-46,reports a study of three ipilimumab-treated patients that had beenprevaccinated with either: gp100 DNA; a gp100²⁰⁹⁻²¹⁷ andtyrosinase³⁶⁹⁻³⁷⁷ peptide vaccine plus GM-CSF DNA; or recombinant humanNY-ESO-1 protein. In patient IMF-11, who had been prevaccinated withrecombinant human NY-ESO-1 protein, subsequent in vitro immunomonitoringwas performed with 20-mer NY-ESO-1 overlapping peptides; however, thesepeptides were not used in the vaccine itself. The time from vaccinationto ipilimumab treatment ranged from 10 months to 2.5 years. Thereremains a need to provide methods and compositions that provide clinicalbenefit in humans across a broad range of patients.

WO 2007/113648 relates to uses and compositions comprising ananti-CTLA-4 antibody and at least one therapeutic agent for thetreatment of cancer. The combination of an anti-CTLA-4 antibody,CP-675,206, and (whole) tumour antigen is mentioned but there are noexperimental data on this combination. For instance, Example 15 relatesto administration of an influenza virus vaccine and the CP-675,206antibody in Rhesus monkeys but no data are provided on theadministration of a cancer vaccine derived from a self-antigen incombination with an immune checkpoint inhibitor. This is significant,given the preponderance of evidence indicating that the administrationof an immune checkpoint inhibitor in combination with a peptide-basedcancer vaccine does not increase immune responses or result in improvedclinical efficacy in humans, as explained above.

Foy et al. Cancer Immunol Immunother. 2016 May; 65(5):537-49, relates tothe use of MVA-BN-HER2 poxvirus-based active immunotherapy alone or incombination with CTLA-4 checkpoint blockade in a therapeutic CT26-HER-2lung metastasis mouse model. MVA-BN-HER2 is a modified vacciniaAnkara-based recombinant vector that encodes a modified form of thehuman epidermal growth factor receptor 2 (HER-2). The Foy et al. studywas performed in mice, where human HER-2 is not a self-antigen. Asdiscussed above in the context of peptide-based cancer vaccines incombination with CTLA-4 blockade, there is a concern that experiments inmice do not necessarily translate into increased immune responses andimproved clinical efficacy in humans.

Zanetti Nat Rev Clin Oncol. 2017 February; 14(2):115-128 is aPerspectives opinion article on telomerase reverse transcriptase inanticancer immunotherapy. The discussion encompasses immune checkpointinhibitors; in particular, in the context of the tumour microenvironmentand its role in determining the success of therapeutic vaccination (FIG.1). In particular, the role of immune checkpoint inhibitors in releasingthe brake on (pre-existing) naturally acquired immune responses isdiscussed and the ability of immune checkpoint inhibitors to restore theactivity of exhausted T cells. However, no experimental data areprovided to support the discussion which, as mentioned above, issignificant given the preponderance of evidence indicating that theadministration of an immune checkpoint inhibitor in combination with apeptide-based cancer vaccine does not increase immune responses orresult in improved clinical efficacy in humans.

WO 03/086459 relates to methods of promoting or potentiating a secondaryor memory immune response using anti-CTLA-4 antibodies. Example 1relates to a melanoma cell vaccine eliciting a CD4+ and CD8+ response inwhich a whole cell vaccine expressing GM-CSF was used in Cynomolgusmonkeys. Example 5 relates to administration of an anti-CTLA-4 antibodyin conjunction with vaccination with two HLA-A*0201-restricted gp100peptides in humans. These peptides were 9 amino acids in length and werethe same peptides (i.e. gp100:209-217(210M) and gp100:280-288(288V))that were used in the cancer vaccine of Hodi et al. 2010 and whichproduced no additional clinical benefit in humans when combined withipilimumab.

WO 2011/101173 discloses various polypeptides from human telomerasereverse transcriptase (hTERT) for the treatment of cancer. There is nodisclosure of immune checkpoint inhibitors. There remains a need toprovide further anti-cancer treatments.

The present invention seeks to alleviate the at least some of the aboveproblems and, in some aspects, seeks to provide a peptide-based cancervaccine with broad population coverage that improves clinical responserates in cancer patients when combined with a checkpoint inhibitor.

In this regard it is to be noted that MHC class I molecules are found onthe surface of most cells and typically bind polypeptides which arebetween 8 and 10 amino acid residues in length. MHC class I moleculespresent polypeptides, which are derived from cytosolic proteins byproteolysis, to CD8+ T cells (also known as cytotoxic T cells or CTLs)in order to elicit a CD8+ T cell response. In contrast, MHC class IImolecules are found on the surface of antigen presenting cells and bindpolypeptides that are generally longer, typically between 12 and 24amino acids in length. MHC class II molecules present polypeptides,which are derived from extracellular proteins that have beeninternalised by endocytosis and digested, to CD4+ T cells (otherwiseknown as helper T cells or Th cells) in order to elicit a CD4+ T cellresponse.

The present inventors have made the observation that in the studiesdescribed in Hodi et al. 2006 and Sarnaik et al. 2011, the peptide-basedcancer vaccines comprised short (9-mer) peptides, designed to elicitcytotoxic T cell responses in patients positive for HLA-A2. The presentinvention arises out of the surprising finding that the combination of aCTLA-4 inhibitor and a peptide-based cancer vaccine comprising at leastone peptide that is 12 amino acids or longer (i.e. a “long” peptide) andwhich is capable of inducing a helper T cell response produces asynergistic effect in the treatment of cancer. This finding led to thesurprising realisation that a peptide-based cancer vaccine comprising atleast one long peptide of a self-antigen, which is capable of elicitinga helper T cell response in a broad range of patients, in combinationwith an immune checkpoint inhibitor could result in improved immuneresponses and improved clinical efficacy in the treatment of canceracross a broad range of the population.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention, there is provideda polypeptide for use in medicine wherein the polypeptide isadministered simultaneously, separately or sequentially with an immunecheckpoint inhibitor, and

wherein the polypeptide comprises at least one polypeptide comprising aregion of at least 12 amino acids of a self-antigen or a sequence havingat least 80% identity to the region.

Preferably, the polypeptide is less than 100 amino acids in length.

In particular, the polypeptide elicits a CD4+ T-cell response.

According to a second aspect of the present invention, there is provideda nucleic acid molecule for use in medicine wherein the nucleic acidmolecule is administered simultaneously, separately or sequentially withan immune checkpoint inhibitor, and wherein the nucleic acid moleculecomprises a nucleotide sequence encoding at least one polypeptidecomprising a region of at least 12 amino acids of a self-antigen or asequence having at least 80% identity to the region.

According to a third aspect of the present invention, there is provideda T-cell receptor, or a T-cell displaying the T-cell receptor, for usein medicine wherein the T-cell receptor or T-cell is administeredsimultaneously, separately or sequentially with an immune checkpointinhibitor, and

wherein the T-cell receptor or T-cell is specific for a polypeptideconsisting of at least 12 amino acids of a self-antigen, or a sequencehaving at least 80% identity to the polypeptide, when the polypeptide ispresented on an MHC molecule.

According to a fourth aspect of the present invention, there is providedan immune checkpoint inhibitor for use in medicine wherein the immunecheckpoint inhibitor is administered simultaneously, separately orsequentially with:

-   -   i) a polypeptide comprising at least one polypeptide comprising        a region of at least 12 amino acids of a self-antigen or a        sequence having at least 80% identity to the region;    -   ii) a nucleic acid molecule comprising a nucleotide sequence        encoding at least one polypeptide comprising a region of at        least 12 amino acids of a self-antigen or a sequence having at        least 80% identity to the region;    -   iii) a T-cell receptor specific for a polypeptide consisting of        at least 12 amino acids of a self-antigen, or a sequence having        at least 80% identity to the polypeptide, when the polypeptide        is presented on an MHC molecule; or    -   iv) a T-cell displaying a T-cell receptor as defined in iii).

Conveniently, the polypeptide under item i) is less than 100 amino acidsin length.

Preferably, the polypeptide of the invention, the nucleic acid moleculeof the invention, the T-cell or T-cell receptor of the invention or theimmune checkpoint inhibitor of the invention is for use in the treatmentof cancer.

Preferably, the polypeptide of the invention, the nucleic acid moleculeof the invention, the T-cell or T-cell receptor of the invention or theimmune checkpoint inhibitor of the invention is for use in thevaccination for cancer.

According to a fifth aspect of the present invention, there is provideda method of treatment of cancer in a patient, comprising the steps of:

-   -   i) inhibiting an immune checkpoint; and    -   ii) simultaneously, separately or sequentially administering:        -   a) at least one polypeptide comprising a region of at least            12 amino acids of a self-antigen or a sequence having at            least 80% identity to the region;        -   b) at least one nucleic acid molecule comprising a            nucleotide sequence encoding at least one polypeptide            comprising a region of at least 12 amino acids of a            self-antigen or a sequence having at least 80% identity to            the region;        -   c) a T-cell receptor specific for a polypeptide consisting            of at least 12 amino acids of a self-antigen, or a sequence            having at least 80% identity to the polypeptide, when the            polypeptide is presented on an MHC molecule; or        -   d) a T-cell displaying a T-cell receptor as defined in c).

Advantageously, the at least one polypeptide under item a) is less than100 amino acids in length.

Conveniently, there is provided a method of vaccination for cancer in apatient as set out in the fifth aspect of the invention.

Preferably, the at least one polypeptide, the T-cell or T-cell receptorin combination with the immune checkpoint inhibitor or the inhibition ofthe immune checkpoint produce a synergistic effect in the treatment ofcancer.

Conveniently, the at least one nucleic acid molecule in combination withthe immune checkpoint inhibitor or the inhibition of the immunecheckpoint produces a synergistic effect in the treatment of cancer.

Preferably, the at least one polypeptide, the at least one nucleic acidmolecule, the T-cell or T-cell receptor in combination with the immunecheckpoint inhibitor or the inhibition of the immune checkpoint producea synergistic effect in the vaccination for cancer.

Advantageously, the polypeptide, the nucleic acid molecule, the T-cellor T-cell receptor in combination with the immune checkpoint inhibitorare for use in generating an accelerated CD4+ T cell immune response.

According to a sixth aspect the present invention, there is provided acomposition or kit suitable for the treatment of cancer, comprising:

-   -   i) a) at least one polypeptide comprising a region of at least        12 amino acids of a self-antigen or a sequence having at least        80% identity to the region;        -   b) at least one nucleic acid molecule comprising a            nucleotide sequence encoding at least one polypeptide            comprising a region of at least 12 amino acids of a            self-antigen or a sequence having at least 80% identity to            the region;        -   c) a T-cell receptor specific for a polypeptide consisting            of at least 12 amino acids of a self-antigen, or a sequence            having at least 80% identity to the polypeptide, when the            polypeptide is presented on an MHC molecule; or        -   d) a T-cell displaying a T-cell receptor as defined in c)            and    -   ii) an immune checkpoint inhibitor,        wherein the at least one polypeptide, the T-cell or T-cell        receptor in combination with the immune checkpoint inhibitor        produce a synergistic effect in the treatment of cancer.

Preferably, wherein the at least one nucleic acid molecule incombination with the immune checkpoint inhibitor produces a synergisticeffect in the treatment of cancer.

Preferably, the at least one polypeptide, the at least one nucleic acidmolecule, the T-cell or T-cell receptor in combination with the immunecheckpoint inhibitor produce a synergistic effect in the vaccination forcancer.

Conveniently, the at least one polypeptide under item a) is less than100 amino acids in length.

Preferably, the at least one polypeptide comprises a region of at least15, 20, 25 or 30 amino acids of a self-antigen or a sequence having atleast 80% identity to the region.

Preferably, the polypeptide comprises a region of at least 15, 20, 25 or30 amino acids of a self-antigen or a sequence having at least 80%identity to the region.

Conveniently, the self-antigen is a universal tumour antigen, preferablytelomerase reverse transcriptase, Top2alpha, survivin or CYP1B1.

Advantageously, the self-antigen is telomerase reverse transcriptase andwherein the at least one polypeptide comprises a polypeptide comprisinga sequence of SEQ ID NO. 1 or a sequence having at least 80% sequenceidentity thereto or an immunogenic fragment thereof comprising at least12 amino acids.

Conveniently, the self-antigen is telomerase reverse transcriptase andthe or the at least one polypeptide comprises:

-   -   i) a polypeptide comprising a sequence of SEQ ID NO. 1;    -   ii) an immunogenic fragment of i) comprising at least 12 amino        acids; or    -   iii) a sequence having at least 80% sequence identity to i) or        ii).

According to a seventh aspect of the present invention, there isprovided a composition or kit suitable for the treatment of cancer,comprising:

-   -   i) at least one polypeptide, wherein the at least one        polypeptide comprises a polypeptide comprising a sequence of        SEQ. ID NO. 1 or a sequence having at least 80% sequence        identity thereto or an immunogenic fragment thereof comprising        at least 12 amino acids; and    -   ii) an immune checkpoint inhibitor.

Conveniently, the at least one polypeptide under item i) comprises:

-   -   a) a polypeptide comprising a sequence of SEQ ID NO. 1;    -   b) an immunogenic fragment of a) comprising at least 12 amino        acids; or    -   c) a sequence having at least 80% sequence identity to a) or b).

Preferably, the at least one polypeptide is a cocktail of polypeptidesand wherein the cocktail of polypeptides further comprises:

-   -   a polypeptide comprising a sequence of SEQ. ID NO. 2 or a        sequence having at least 80% sequence identity thereto or an        immunogenic fragment thereof comprising at least 12 amino acids;        and optionally,    -   a polypeptide comprising a sequence of SEQ. ID NO. 3 or a        sequence having at least 80% sequence identity thereto or an        immunogenic fragment thereof comprising at least 12 amino acids.

Conveniently, the or the at least one polypeptide is a cocktail ofpolypeptides and wherein the cocktail of polypeptides further comprises:

-   -   a polypeptide comprising:        -   a) a sequence of SEQ. ID NO. 2;        -   b) an immunogenic fragment of a) comprising at least 12            amino acids; or        -   c) a sequence having at least 80% sequence identity to a) or            b), and optionally,    -   a polypeptide comprising:        -   a) a sequence of SEQ. ID NO. 3;        -   b) an immunogenic fragment of a) comprising at least 12            amino acids; or        -   c) a sequence having at least 80% sequence identity to a) or            b).

According to an eighth aspect of the present invention, there isprovided a composition or kit suitable for the treatment of cancer,comprising:

-   -   i) at least one nucleic acid molecule, wherein the at least one        nucleic acid molecule comprises a nucleic acid sequence encoding        a polypeptide comprising a primary sequence of SEQ. ID NO. 1 or        a secondary sequence having at least 80% sequence identity to        the primary sequence or an immunogenic fragment of the primary        sequence or the secondary sequence comprising at least 12 amino        acids; and    -   ii) an immune checkpoint inhibitor.

Advantageously, the at least one nucleic acid molecule is a cocktail ofnucleic acid molecules, and wherein the cocktail of nucleic acidmolecules further comprises:

-   -   a nucleic acid molecule comprising a nucleic acid sequence        encoding a polypeptide comprising a primary sequence of SEQ. ID        NO. 2 or a secondary sequence having at least 80% sequence        identity to the primary sequence or an immunogenic fragment of        the primary sequence or the secondary sequence comprising at        least 12 amino acids; and optionally,    -   a nucleic acid molecule comprising a nucleic acid sequence        encoding a polypeptide comprising a primary sequence of SEQ. ID        NO. 3 or a secondary sequence having at least 80% sequence        identity to the primary sequence or an immunogenic fragment of        the primary sequence or the secondary sequence comprising at        least 12 amino acids.

According to a ninth aspect of the present invention, there is provideda composition or kit suitable for the treatment of cancer, comprising:

-   -   i) at least one T-cell receptor, or at least one T-cell        displaying the T-cell receptor, wherein the T-cell receptor or        T-cell is specific for a polypeptide consisting of SEQ. ID NO.        1, or a sequence having at least 80% identity to the        polypeptide, when the polypeptide is presented on an MHC        molecule; and    -   ii) an immune checkpoint inhibitor.

Conveniently, the polypeptide under item i) consists of:

-   -   a) a sequence of SEQ ID NO. 1;    -   b) an immunogenic fragment of a) comprising at least 12 amino        acids; or    -   c) a sequence having at least 80% sequence identity to a) or b),        when the polypeptide is presented on an MHC molecule.

Preferably, the at least one T-cell receptor is a cocktail of T-cellreceptors or the at least one T-cell is a cocktail of T-cells andwherein the cocktail further comprises:

-   -   a T-cell receptor, or a T-cell displaying the T-cell receptor,        specific for a polypeptide consisting of a sequence of SEQ. ID        NO. 2, or a sequence having at least 80% sequence identity        thereto, when the polypeptide is presented on an MHC molecule;        and optionally,    -   a T-cell receptor, or a T-cell displaying the T-cell receptor,        specific for a polypeptide consisting of a sequence of SEQ. ID        NO. 3, or a sequence having at least 80% sequence identity        thereto, when the polypeptide is presented on an MHC molecule.

Preferably, the at least one T-cell receptor is a cocktail of T-cellreceptors or the at least one T-cell is a cocktail of T-cells andwherein the cocktail further comprises:

-   -   a T-cell receptor, or a T-cell displaying the T-cell receptor,        specific for a polypeptide consisting of:        -   a) a sequence of SEQ. ID NO. 2;        -   b) an immunogenic fragment of a) comprising at least 12            amino acids; or        -   c) a sequence having at least 80% sequence identity to a) or            b), when the polypeptide is presented on an MHC molecule;            and optionally,    -   a T-cell receptor, or a T-cell displaying the T-cell receptor,        specific for a polypeptide consisting of:        -   a) a sequence of SEQ. ID NO. 3        -   b) an immunogenic fragment of a) comprising at least 12            amino acids; or        -   c) a sequence having at least 80% sequence identity to a) or            b), when the polypeptide is presented on an MHC molecule.

Conveniently, the composition or kit according to the sixth, seventh,eighth or ninth aspect of the invention is suitable for vaccination forcancer.

Advantageously, the immune checkpoint inhibitor is a CTLA-4 inhibitor, aPD-1 inhibitor or a PD-L1 inhibitor, or wherein the inhibition of theimmune checkpoint is by administration of a CTLA-4 inhibitor, a PD-1inhibitor or a PD-L1 inhibitor.

In particular, the immune checkpoint inhibitor is an inhibitor of amember of the CD28CTLA-4 immunoglobulin superfamily.

Conveniently, the CTLA-4 inhibitor is an anti-CTLA-4 antibody or a smallmolecule CTLA-4 antagonist, wherein the PD-1 inhibitor is an anti-PD-1antibody or a small molecule PD-1 antagonist, or wherein the PD-L1inhibitor is an anti-PD-L1 antibody or a small molecule PD-L1antagonist.

Preferably, the anti-CTLA-4 antibody is: ipilimumab or tremelimumab,wherein the anti-PD-1 antibody is nivolumab or pembrolizumab, or whereinthe anti-PD-L1 antibody is MPDL3280A or BMS-936559.

According to a tenth aspect of present invention, there is provided apharmaceutical composition comprising the composition of the presentinvention and a pharmaceutically acceptable adjuvant, diluent orexcipient and optionally another therapeutic ingredient.

Conveniently, the kit of the present invention, further comprises apharmaceutically acceptable adjuvant, diluent or excipient andoptionally another therapeutic ingredient.

Advantageously, the method of treatment of the present invention,further comprises the administration of a pharmaceutically acceptableadjuvant, diluent or excipient and optionally another therapeuticingredient.

According to an eleventh aspect of the present invention, there isprovided a method of treatment of cancer in a patient comprisingadministering the composition of the present invention or thepharmaceutical composition of the present invention to the patient.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidues is a modified residue, or a non-naturally occurring residue,such as an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

The term “amino acid” as used herein refers to naturally occurring andsynthetic amino acids, as well as amino acid analogues and amino acidmimetics that have a function that is similar to the naturally occurringamino acids. Naturally occurring amino acids are those encoded by thegenetic code, as well as those modified after translation in cells (e.g.hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine). The phrase“amino acid analogue” refers to compounds that have the same basicchemical structure (an alpha carbon bound to a hydrogen, a carboxygroup, an amino group, and an R group) as a naturally occurring aminoacid but have a modified R group or modified backbones (e.g. homoserine,norleucine, methionine sulfoxide, methionine methyl sulphonium). Thephrase “amino acid mimetic” refers to chemical compounds that havedifferent structures from but similar functions to naturally occurringamino acids.

The term “fragment” as used herein in relation to a polypeptide means aconsecutive series of amino acids that form part of the polypeptide. An“immunogenic fragment” of a polypeptide is a fragment as previouslydefined which is capable of eliciting an immune response, such as aT-cell response, when administered to an individual. In some embodimentsan “immunogenic fragment” of a polypeptide is a fragment as previouslydefined which is capable of eliciting an MHC class II restricted immuneresponse.

The terms “gene”, “polynucleotides”, and “nucleic acid molecules” areused interchangeably herein to refer to a polymer of multiplenucleotides. The nucleic acid molecules may comprise naturally occurringnucleic acids or may comprise artificial nucleic acids such as peptidenucleic acids, morpholin and locked nucleic acid as well as glycolnucleic acid and threose nucleic acid.

The term “nucleotide” as used herein refers to naturally occurringnucleotides and synthetic nucleotide analogues that are recognised bycellular enzymes.

The term “cancer” as used herein refers to a group of diseases that arecharacterised by new and abnormal and/or uncontrolled proliferation ofcells in an individual. Cancer cells have the capacity to invadeadjacent tissues and/or to spread to other sites in the body (i.e. thecancer cells are capable of metastasis).

The term “treatment” as used herein refers to any partial or completetreatment and includes: inhibiting the disease or symptom, i.e.arresting its development; and relieving the disease or symptom, i.e.causing regression of the disease or symptom.

The term “self-antigen” as used herein refers to an antigen that isderived from a naturally-occurring protein within the human body. Undernormal conditions, the immune system does not react to self-antigens dueto negative selection of T cells in the thymus. However, in anindividual with cancer, self-antigens may be recognised as foreign bythe immune system (for example, as a result of the cancer celloverexpressing the protein from which the self-antigen is derived orexpressing it inappropriately given the tissue in which the cancerdeveloped) and a T cell immune response is mounted against theself-antigen. In some embodiments, the self-antigen may be referred toas a “tumour-associated antigen” i.e. an antigen associated with acancer cell as well as a normal cell. An example of a self-antigen istelomerase reverse transcriptase.

The term “universal tumour antigen” as used herein refers to an antigenthat is expressed in (nearly) all tumours, such as in at least 80%, 85%or 90% of all tumour types. In some embodiments, the universal tumourantigen is directly involved in the malignant phenotype of the tumour.Examples of a universal tumour antigen include telomerase reversetranscriptase, Top2alpha, survivin and CYP1B1.

The term “T-cell” (also known as “T lymphocyte”) as used herein refersto a cell that is capable of recognising a specific antigen and whichcomprises a cell surface T-cell receptor. The term “T-cell” comprisesdifferent types of T cell, such as: CD4+ T cells (also known as helper Tcells or Th cells), CD8+ T cells (also known as cytotoxic T cells orCTLs), memory T cells and regulatory T cells (Tregs).

The term “the T-cell receptor” as used herein refers to an antigenreceptor of the T-cell. In some embodiments, the T-cell receptorrecognises (i.e. binds to) a polypeptide when presented by an MHCmolecule.

The term “a T-cell displaying the T-cell receptor” as used herein refersto a T-cell that comprises the T-cell receptor on its cell surface. Insome embodiments, the T-cell receptor is responsible for recognising(i.e. binding to) a polypeptide when presented by an MHC molecule. Insome embodiments, the binding of the T-cell receptor to the polypeptidewhen presented by the MHC molecule results in activation of the T-celldisplaying the T-cell receptor. T cell activation can be measured usingT-cell response assays and ELISPOT assays as described herein

The term “the T-cell receptor or T-cell is specific for a polypeptide”as used herein refers to a T-cell receptor or a T cell comprising theT-cell receptor that is capable of recognising (i.e. binding to) thepolypeptide when presented on an MHC molecule. In some embodiments, thepolypeptide to which the T-cell receptor (or the T-cell displaying theT-cell receptor) is specific, is of a length that is longer than thatwhich would normally be accommodated on an MHC molecule. In theseembodiments, the term “the T-cell receptor or T-cell is specific for apolypeptide” as used herein refers to the recognition by the T-cellreceptor or T-cell of an immunogenic fragment of the polypeptide whenpresented on the MHC molecule. In some embodiments, the binding of theT-cell receptor or T-cell to the polypeptide to which it is specificresults in activation of a T-cell. T cell activation can be measuredusing T-cell response assays and ELISPOT assays as described herein.

The term “MHC molecule” as used herein refers to a protein structurewhich assembles with a polypeptide and which is capable of displayingthe polypeptide at a cell surface to a T-cell. MHC molecules are encodedby genes within the major histocompatibility complex. In someembodiments, the term “MHC molecule” refers to an MHC class I moleculesand/or an MHC class II molecule.

The term “immune checkpoint” as used herein refers to any point at whichan immune response is limited. Immune checkpoints are inhibitorypathways that slow down or stop immune reactions and prevent excessivetissue damage from uncontrolled activity of immune cells. Examples of an“immune checkpoint” include the cytotoxic T-lymphocyte-associatedprotein 4 (CTLA-4) checkpoint and the programmed cell death protein 1(PD-1) checkpoint.

The term “immune checkpoint inhibitor” as used herein refers to anycompound, substance or composition (e.g. any small molecule, chemicalcompound, antibody, nucleic acid molecule, polypeptide, or fragmentsthereof, a vaccine or viral vaccine) that is capable of down-regulatingor blocking an immune checkpoint allowing more extensive immuneactivity. The term “checkpoint inhibitor” is used interchangeably hereinwith “immune checkpoint inhibitor”. In some embodiments, the immunecheckpoint inhibitor is an antibody that specifically binds to a proteininvolved in the immune checkpoint pathway thereby disrupting anddown-regulating the overall activity of the immune checkpoint. Examplesof such an immune checkpoint inhibitor include an anti-CTLA-4 antibody(such as ipilimumab, tremelimumab or AGEN-1884) and an anti-PD-1antibody (such as nivolumab or pembrolizumab). In alternativeembodiments, the immune checkpoint inhibitor is a small moleculeantagonist that interferes with and/or inhibits the activity of aprotein involved in the immune checkpoint pathway and therebydown-regulates the overall activity of the immune checkpoint. In apreferred embodiment, the small molecule antagonist targets the CTLA-4and/or PD-1 proteins in order to down-regulate the CTLA-4 and/or PD-1checkpoints (i.e. the small molecule antagonist is a small moleculeCTLA-4 antagonist or a small molecule PD-1 antagonist). In additionalembodiments, the immune checkpoint inhibitor is targeted at anothermember of the CD28CTLA4 Ig superfamily such as BTLA, LAG3, ICOS, PDL1 orKIR (Page et al., Annual Review of Medicine 65:27 (2014)). In furtheradditional embodiments, the immune checkpoint inhibitor is targeted at amember of the TNFR superfamily such as CD40, OX40, CD137, GITR, CD27 orTIM-3. In a further embodiment, the immune checkpoint inhibitor targetsIndoleamine 2,3-dioxygenase (IDO). In some cases targeting an immunecheckpoint is accomplished with an inhibitory antibody or similarmolecule. In other cases, it is accomplished with an agonist for thetarget; examples of this class include the stimulatory targets OX40 andGITR.

In a preferred embodiment, the immune checkpoint inhibitor targets animmune checkpoint that is involved in the regulation of a T-cell. Insome embodiments, the immune checkpoint that is targeted is a negativeregulator of T-cell activity; thus the action of the immune checkpointinhibitor allows for more extensive T-cell activity. As discussed above,in some embodiments, the immune checkpoint inhibitor targets a member ofthe CD28CTLA4 immunoglobulin (Ig) superfamily. Proteins in theimmunoglobulin superfamily possess an immunoglobulin domain (also knownan immunoglobulin fold) which is a characteristic beta-sheet fold.CTLA-4, PD-1 and PD-L1 are examples of members of the CD28CTLA4 Igsuperfamily.

The term “inhibiting an immune checkpoint” as used herein refers todown-regulating or blocking an immune checkpoint in order to allow moreextensive immune activity. In some embodiments, inhibiting an immunecheckpoint is achieved using at least one of the immune checkpointinhibitors described above.

The term “synergistic effect in the treatment of cancer” as used hereinrefers to presence of at least one of the following combination offactors in patients who have been administered a peptide-based (or anucleic acid molecule-based) cancer vaccine and a checkpoint inhibitorin comparison with a control (for example, patients who have beenadministered the peptide-based cancer vaccine without the checkpointinhibitor; or alternatively, patients who have been administered thecheckpoint inhibitor without the peptide-based cancer vaccine).

-   -   1. A reduction in the time required by the immune system of the        patients to mount a measurable immune response to the peptide(s)        of the vaccine. In other words, an accelerated CD4+ T cell        immune response is generated.    -   2. The mounting of a strong immune response to the peptide(s) of        the vaccine by the patients. In one embodiment, a “strong immune        response” as used herein refers to, when across an average of 10        patients, the mean peak immune response is an SI of at least 17,        preferably at least 19.    -   3. An improved clinical outcome in the patients.

In some embodiments, the term “synergistic effect in the treatment ofcancer” refers to the presence of at least two of said factors or allthree of said factors in patients. In one embodiment, an additionalfactor, namely, the induction of a broad immune response (i.e. themounting of an immune response against 2, 3 or more vaccine components),is further evidence of a synergistic effect in the treatment of cancer.In a preferred embodiment, immune responses are measured by a T cellresponse assay (proliferation by 3H-thymidine incorporation) usingpatient blood samples as explained in the Materials and Methods sectionherein. A specific T-cell response is considered positive if the peptideresponse is at least 3 times the background (Stimulation Index, SI≥3).In one embodiment, a synergistic effect is provided when, across anaverage of ten patients, over 50% exhibit a positive immune response 7weeks after the first administration of the peptide vaccine; and themean peak immune response is an SI of at least 17, preferably at least19. In some embodiments, an improved clinical outcome is a partial orcomplete response (also known as partial or complete remission) orstable disease. A complete response refers to the disappearance ofdetectable tumour or cancer in the body in response to treatment; apartial response refers to a decrease in tumour size, or in the extentof cancer in the body, in response to treatment; and stable diseasemeans that tumour or cancer in the body is neither decreasing norincreasing in extent or severity.

The term “generating an accelerated CD4+ T cell immune response” as usedherein refers to a reduction in the amount of time required by theimmune system to mount a measurable CD4+ T cell immune response. In oneembodiment, a response time refers to the time from: the start ofvaccination; to: the expansion of vaccine specific CD4+ T-cells to alevel defining a positive vaccine response. In this embodiment, anaccelerated CD4+ T cell response is defined as T2<T1 where T1 is theresponse time of the vaccine alone and T2 is the response time of thecombined treatment of the vaccine and the immune checkpoint inhibitor.In one embodiment, the vaccine comprises a polypeptide of the invention;in an alternative embodiment, the vaccine comprises a nucleic acidmolecule of the invention.

In certain embodiments, where the vaccine is a clinical vaccine, T1 andT2 refer to the average values in a treated population. In oneembodiment, T1 and T2 refer to the average values across 10 or morepatients. The level defining a positive immune response is dependent onthe assay used. In one embodiment, it is based on a detection threshold;in an alternative embodiment, it is a pre-defined value. In certainembodiments, the assay used to measure the immune response is a T cellproliferation assay (proliferation by 3H-thymidine incorporation) asdescribed herein. In one embodiment, the level defining a positiveimmune response is pre-defined to a stimulation index (SI) of 3 (SI≥3).It is to be understood that this level is higher than the detectionthreshold and is selected, in certain embodiments, to represent apotentially clinically relevant immune response. In other embodiments,the SI is less than or higher than 3. In one embodiment, the SI is 2 or4.

In one embodiment, T1 as defined above is the number of weeks to when50% or more of patients treated with the vaccine alone have a positiveimmune response; and T2 is the number of weeks to when 50% or more ofpatients treated with the combination of the vaccine and the immunecheckpoint inhibitor have a positive immune response. In one embodiment,an accelerated immune response refers to a 60% decrease in T2 comparedwith T1 (for example, T2 is 4 weeks as compared with T1 which is 10weeks). In other embodiments, an accelerated CD4+ immune response refersto a 55%, 50%, 45%, 40%, 35% or 30% decrease in T2 as compared with T1.Samples are collected at discrete time points and so, in someembodiments, calculation of T1 and T2 requires interpolation.

The term “telomerase reverse transcriptase” (TERT) as used herein refersto the catalytic component of the telomerase holoenzyme complex whosemain activity is the elongation of telomeres by acting as a reversetranscriptase that adds simple sequence repeats to chromosome ends bycopying a template sequence within the RNA component of the telomeraseenzyme. In some embodiments, the term telomerase refers to the humantelomerase reverse transcriptase protein (hTERT). The full-length hTERTsequence is set out in GenBank accession no. AF015950.1 and is set forthin SEQ ID NO. 6.

In this specification, the percentage “identity” between two sequencesis determined using the BLASTP algorithm version 2.2.2 (Altschul,Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang,Zheng Zhang, Webb Miller, and David J. Lipman (1997), “Gapped BLAST andPSI-BLAST: a new generation of protein database search programs”,Nucleic Acids Res. 25:3389-3402) using default parameters. Inparticular, the BLAST algorithm can be accessed on the internet usingthe URL http://www.ncbi.nlm.nih.gov/blast/.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic showing the mechanism by which an embodiment ofthe present invention elicits an immune response.

FIGS. 2A and 2B are bar graphs summarising T-cell responses detected ina melanoma patient vaccinated with a combination of SEQ. ID NOS. 1, 2and 3 using a T cell proliferation assay and an ELISPOT assayrespectively. CD4+ T-cell responses against SEQ. ID NOS. 1 and 2 as wellas the combination of SEQ ID NOS. 1, 2 and 3 were detected.Proliferation in response to peptide-loaded PBMC was measured by3H-thymidine incorporation. A stimulation index of ≥3 is considered animmune response. 719-20 refers to SEQ. ID NO: 1, 725 refers to SEQ. IDNO. 2, 728 refers to SEQ. ID NO. 3, and hTERT1 mix refers to acombination of SEQ. ID NOS. 1, 2 and 3.

FIGS. 3A-C are bar graphs summarising CD4+ T-cell responses detected inmelanoma patients and a lung cancer patient against polypeptides havinga sequence of SEQ ID NO. 1 and fragments thereof. Proliferation inresponse to peptide-loaded PBMC was measured by 3H-thymidineincorporation. A stimulation index of ≥2 is considered an immuneresponse. 719-20-13 to 719-20-16 and 719-20-2 to 719-20-9 refer tofragments of SEQ ID NO. 1 comprising 14 amino acids thereof.

FIG. 4 is a bar graph summarising CD4+ T-cell responses detected in amelanoma patient and an ovarian cancer patient against polypeptideshaving a sequence of SEQ ID NO. 2 and fragments thereof. Proliferationin response to peptide-loaded PBMC was measured by 3H-thymidineincorporation. A stimulation index of ≥2 is considered an immuneresponse. 725-1 to 725-4 refer to fragments of SEQ ID NO. 2 comprising12 amino acids thereof.

FIG. 5 is a bar graph summarising CD4+ T-cell responses detected in apancreatic cancer patient and a glioblastoma patient againstpolypeptides having a sequence of SEQ ID No. 3 and fragments thereof.Proliferation in response to peptide-loaded

PBMC was measured by 3H-thymidine incorporation. A stimulation index of≥3 is considered an immune response. 728-1 to 728-4 refer to fragmentsof SEQ ID NO. 3 comprising 12 amino acids thereof.

FIG. 6 is a bar graph summarising CD4+ T-cell responses detected in acancer patient with prostate cancer vaccinated with a combination ofSEQ. ID NOS. 1, 2 and 3. CD4+ T-cell responses against overlapping14-mer peptides from SEQ. ID NO. 1 were detected following vaccinationand responding T cells cloned. The data in FIG. 6 indicate proliferativeresponses of selected CD4+ T cell clones. Proliferation in response topeptide-loaded PBMC was measured by 3H-thymidine incorporation. Astimulation index of ≥3 is considered an immune response.

FIG. 7 is a graph summarising positive immune responses detected insamples from lung and prostate cancer patients vaccinated with SEQ IDNOS: 1, 2 and 3 and GM-CSF and in samples from melanoma patientsreceiving ipilimumab treatment in combination with vaccination with SEQID NOS: 1, 2 and 3 and GM-CSF. T cell proliferation was measured by3H-thymidine incorporation. A stimulation index of was considered apositive immune response.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a kit for the treatment of cancer. Thekit comprises two components. In a first embodiment, the first componentis at least one polypeptide of a self-antigen, wherein the polypeptideis at least 12 amino acids in length. The second component is an immunecheckpoint inhibitor.

Polypeptides

The first component of the kit for the treatment of cancer is at leastone polypeptide of a self-antigen.

A self-antigen is an antigen that is derived from a naturally-occurringprotein within the human body. Cancer cells may express certainself-antigens at a higher level than normal cells or the self-antigenmay be expressed inappropriately given the tissue in which the cancercell developed. These self-antigens can be regarded as“tumour-associated antigens” and thus represent a potential target forcancer therapy. It is preferred that the self-antigen is a universaltumour antigen, which is an antigen expressed in (nearly) all humantumours. It is to be appreciated that certain tumour associated antigensare both self-antigens and universal tumour antigens. Cancer is aheterogeneous disease and there is high degree of diversity betweendifferent types of cancer as well as between individuals with the sametype of cancer. By targeting universal tumour antigens, theapplicability of the cancer therapy is improved across the patientpopulation (i.e. within and between cancer types).

In a first embodiment of the invention, the self-antigen is thetelomerase reverse transcriptase subunit (“TERT” or “hTERT” for humans)of the telomerase enzyme. The telomerase enzyme is a “self-protein”,that is to say, it is a naturally-occurring protein in the human body.Furthermore, it has been observed that the telomerase enzyme isactivated in the vast majority of all human tumours. In view of this,polypeptides of hTERT are regarded as both self-antigens and universaltumour antigens.

Telomerase is an enzyme that has the function of replicating the 3′ endof the telomere regions of linear DNA strands in eukaryotic cells asthese regions cannot be extended by the enzyme DNA polymerase in thenormal way. The telomerase enzyme comprises a telomerase reversetranscriptase subunit (“TERT” or “hTERT” for humans) and telomerase RNA.By using the telomerase RNA as a template, the telomerase reversetranscriptase subunit adds a repeating sequence to the 3′ end ofchromosomes in eukaryotic cells in order to extend the 3′ end of the DNAstrand. The full-length hTERT sequence is set out in GenBank accessionno. AF015950.1 and is set forth in SEQ ID NO. 6.

Telomerase is expressed in certain normal tissue such as stem cells inthe bone marrow and gastrointestinal tract. However, it has beenobserved that the telomerase enzyme is activated in the vast majority ofall human tumours (for example, Kim et al., Science. 1994266(5193):2011-5; Shay & Wright, FEBS Lett. 2010 584(17):3819-25). It isbelieved that telomerase is activated in the vast majority of humantumours because, without the expression of the telomerase enzyme, thetelomeres of cells are gradually lost, and the integrity of thechromosomes decline with each round of cell division of a cell, whichultimately results in apoptosis of the cells. Thus, expression of thetelomerase enzyme is generally necessary for a cancer cell to developbecause without such expression, programmed cell death will usuallyoccur by default. In view of the role of telomerase activation incancer, polypeptides from hTERT are regarded as universal tumourantigens.

In alternative embodiments, the self-antigen and/or universal tumourantigen is from a protein other than hTERT. In one embodiment, theself-antigen and/or universal tumour antigen is selected from:topoisomerase II alpha (Top2alpha), survivin or cytochrome P450 1B1(CYP1B1) (Park et al., Cancer Immunol Immunother. 2010 (5):747-57;Sørensen et al., Cancer Biol Ther. 2008 7(12):1885-7; Wobser et al.,Cancer Immunol Immunother. 2006 55(10):1294-8; Gribben et al., ClinCancer Res. 2005 11(12):4430-6). In some embodiments, the at least onepolypeptide is a cocktail (i.e. a mixture) of polypeptides. In the firstembodiment, the cocktail of polypeptides comprises at least twodifferent polypeptides of the hTERT protein. However, in someembodiments, the cocktail of polypeptides comprises at least twodifferent polypeptides selected from any one of the differentself-antigens and/or universal tumour antigens. In one embodiment, thecocktail of polypeptides comprises at least two different polypeptidesselected from any one of: hTERT, Top2alpha, survivin or CYP1B1.

The at least one polypeptide of a self-antigen in the first component ofthe kit for the treatment of cancer is at least 12 amino acids inlength.

It is to be appreciated that different lengths of polypeptide elicitdifferent T cell responses. More specifically, in order to elicit a CD8+T-cell response, the polypeptide must be presented on MHC class Imolecules which will typically only bind polypeptides which are between8 and 10 amino acid residues in length. On the other hand, in order toelicit a CD4+ T-cell response, it is necessary for the polypeptide to bepresented on an MHC class II molecule for which the polypeptides maygenerally be longer, typically between 12 and 24 amino acid residues inlength. Therefore, the at least one polypeptide of a self-antigen oruniversal tumour antigen is capable of eliciting a CD4+ T-cell response(i.e. a helper T cell response) because it is of a longer length (i.e.at least 12 amino acids in length).

It is preferred that the at least one polypeptide of the self-antigen isequal to or at least 15 amino acids in length. In some embodiments, theat least one polypeptide of the self-antigen is equal to or at least 16,17, 18, 19, 20, 25 or 30 amino acids in length. In some embodiments, theat least one polypeptide is less than 100 amino acids in lengthpreferably less than 50, 40 or 30 amino acids in length.

In embodiments where the self-antigen is telomerase (more specifically,hTERT), it is preferred that the polypeptide comprises sequences fromSEQ. ID NOS. 1 to 5. It is particularly preferred that the polypeptidecomprises the sequence of SEQ. ID NOS. 1, 2 or 3. It is especiallypreferred that the polypeptide consists of the sequence of SEQ. ID NOS.1, 2 or 3. It is to be understood that such polypeptides are capable ofeliciting a CD4+ T-cell response (i.e. a helper T cell response) becauseeach of the polypeptides is at least 12 amino acids in length. SEQ. IDNO: 1 is 30 amino acids in length; SEQ. ID NOS: 2, 3 and 4 are 15 aminoacids; and SEQ ID NO: 5 is 16 amino acids in length.

In other embodiments, there are provided immunogenic fragments of theaforementioned polypeptides, which comprise at least 12 amino acids ofSEQ. ID NOS: 1 to 5. In one embodiment, the immunogenic fragmentscomprise at least 12, 13 or 14 amino acids of SEQ. ID NOS. 1 to 5. Inanother embodiment, the immunogenic fragments comprise at least 15, 16,17, 18, 19, 20 or 25 amino acids of SEQ. ID NO. 1. In certainembodiments, the cocktail of polypeptides comprises immunogenicfragments of SEQ. ID NOS. 1 to 5, wherein the immunogenic fragmentscomprise at least 12 amino acids. Exemplary immunogenic fragmentsinclude those set out in SEQ ID NOS. 7 to 38. It is to be appreciatedthat the polypeptides of SEQ. ID NOS. 7 to 23 and 24 to 30 are allimmunogenic fragments of the polypeptide of SEQ. ID NO. 1. Thepolypeptides of SEQ. ID NOS. 31 to 34 are all immunogenic fragments ofthe polypeptide of SEQ. ID NO. 2. The polypeptides of SEQ. ID NOS. 35 to38 are all immunogenic fragments of the polypeptide of SEQ. ID NO. 3.

In further embodiments, the at least one polypeptide provided does nothave exact sequence identity to one of the aforementioned polypeptides.Instead, the polypeptide has at least 80% sequence identity to thepolypeptide set out above. It is particularly preferred that thesequence has at least 90%, 95% or 99% sequence identity to that set outabove. It is also preferred that any addition or substitution of aminoacid sequence results in the conservation of the properties of theoriginal amino acid side chain. That is to say the substitution ormodification is “conservative”.

Conservative substitution tables providing functionally similar aminoacids are well known in the art. Examples of properties of amino acidside chains are hydrophobic amino acids (A, I, L, M, F, P, W, Y, V),hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and sidechains having the following functional groups or characteristics incommon: an aliphatic side-chain (G, A, V, L, I, P); a hydroxyl groupcontaining side chain (S, T, Y); a sulphur atom containing side-chain(C, M); a carboxylic acid and amide containing side-chain (D, N, E, Q);a base containing side-chain (R, K, H); and an aromatic containingside-chain (H, F, Y, W). In addition, the following eight groups eachcontain amino acids that are conservative substitutions for one another(see e.g.

Creighton, Proteins (1984):

-   -   1) Alanine (A), Glycine (G);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q)    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);    -   7) Serine (S), Threonine (T); and    -   8) Cysteine (C), Methionine (M).

In some embodiments, the sequence of the at least one polypeptide isaltered in order to change (e.g. increase) the binding affinity of apolypeptide to an MHC class II molecule of a particular HLA allele. Inother embodiments, the polypeptide has further amino acids, in additionto those set out above, at the N- and/or C-terminal thereof. Suchadditional amino acids can also be used to alter (e.g. increase) thebinding affinity of a polypeptide to an MHC molecule.

It is to be understood that the polypeptide is not limited to having asequence corresponding to a fragment of the self-antigen. That is tosay, in some embodiments, the polypeptide comprises additional aminoacid sequences at the N-terminal and/or C-terminal, in addition to theregion corresponding to the self-antigen. However, the regioncorresponding to the self-antigen (i.e. at least 80%, 90%, 95% or 99%identical to as set out above) is at least 12 amino acids in length.

In some further embodiments of the present invention, the at least onepolypeptide is linked (e.g. covalently) to other substances, whileretaining its capability of inducing a CD4+ T-cell response. Such othersubstances include lipids, sugar and sugar chains, acetyl groups,natural and synthetic polymers and the like. The at least onepolypeptide, in certain embodiments, contains modifications suchglycosylation, side chain oxidation or phosphorylation.

In some embodiments, the at least one polypeptide is a cocktail ofpolypeptides, such as a cocktail of polypeptides from the sameself-antigen or from two or more different self-antigens. In oneembodiment, the cocktail comprises at least 2 or at least 3 differentpolypeptides of the self-antigen. It is particularly preferred that inthe cocktail of polypeptides, the polypeptides in the cocktail arecapable of being bound by MHC class II molecules of more than one HLAallele. It is also to be understood that in some embodiments thecocktail comprises more than two polypeptides having different sequences(e.g. 3, 4 or 5 polypeptides).

It is preferred that the cocktail of polypeptides comprises polypeptidesof the hTERT protein. It is preferred that the polypeptides in thecocktail comprise sequences from at least 2 different polypeptidescomprising sequences from SEQ. ID NOS. 1 to 5. It is particularlypreferred that the polypeptides in the cocktail comprise the sequence ofSEQ. ID NOS. 1, 2 and 3. It is especially preferred that thepolypeptides in the cocktail consist of the sequences of SEQ. ID NOS. 1,2 and 3.

In some embodiments, the at least one polypeptide is produced byconventional processes known in the art. Alternatively, the at least onepolypeptide is a fragment of a protein produced by cleavage, forexample, using cyanogen bromide, and subsequent purification. Enzymaticcleavage may also be used. In further embodiments, the at least onepolypeptide is in the form of a recombinant expressed polypeptide. Forexample, a suitable vector comprising a polynucleotide encoding thepolypeptide in an expressible form (e.g. downstream of a regulatorysequence corresponding to a promoter sequence) is prepared andtransformed into a suitable host cell. The host cell is then cultured toproduce the polypeptide of interest. In other embodiments, the at leastone polypeptide is produced in vitro using in vitro translation systems.

Nucleic Acid Molecules

In a second embodiment of the present invention, there is provided anucleic acid molecule comprising a nucleotide sequence encoding apolypeptide as set out above.

In embodiments where the self-antigen is telomerase, it is preferredthat the nucleic acid molecule comprises a nucleotide sequence encodinga polypeptide comprising sequences from SEQ. ID NOS. 1 to 5. It isparticularly preferred that the nucleic acid molecule comprises anucleotide sequence encoding a polypeptide comprising the sequence ofSEQ. ID NOS. 1, 2 or 3. It is especially preferred that the nucleic acidmolecule comprises a nucleotide sequence encoding a polypeptideconsisting of the sequence of SEQ. ID NOS. 1, 2 or 3.

In some embodiments, there is provided a cocktail (that is to say amixture) of nucleic acid molecules such as a cocktail of nucleic acidmolecules comprising nucleotide sequences encoding polypeptides from thesame self-antigen or from two or more different self-antigens. In oneembodiment, the cocktail comprises at least 2 or at least 3 differentnucleic acid molecules comprising nucleotide sequences encodingpolypeptides of the self-antigen. It is particularly preferred that inthe cocktail of nucleic acid molecules, the encoded polypeptides arecapable of being bound by MHC class II molecules of more than one HLAallele. It is also to be understood that in some embodiments thecocktail comprises more than two nucleic acid molecules encodingdifferent polypeptide sequences (e.g. 3, 4 or 5 nucleic acid molecules).

It is preferred that the cocktail of nucleic acid molecules comprisenucleotide sequences encoding polypeptides of the hTERT protein. It ispreferred that the encoded polypeptide sequences in the cocktailcomprise sequences from at least 2 different polypeptides comprisingsequences from SEQ. ID NOS. 1 to 5. It is particularly preferred thatthe encoded polypeptides in the cocktail comprise the sequence of SEQ.ID NOS. 1, 2 and 3. It is especially preferred that the encodedpolypeptides in the cocktail consist of the sequences of SEQ. ID NOS. 1,2 and 3.

In alternative variants, the sequence of the encoded polypeptide is notidentical to that aforementioned but instead has at least 80%, 90%, 95%or 99% sequence identity thereto. In any case, the encoded polypeptideis less than 100 amino acids in length preferably less than 50, 40 or 30amino acids in length.

In some further embodiments of the present invention, the or eachnucleic acid molecule is linked (e.g. covalently) to other substances.

It is to be appreciated that, owing to the degeneracy of the geneticcode, nucleic acid molecules encoding a particular polypeptide may havea range of polynucleotide sequences. For example, the codons GCA, GCC,GCG and GCT all encode the amino acid alanine.

The nucleic acid molecules may be either DNA or RNA or derivativesthereof.

T-Cell Receptor or T-Cell

In a third embodiment of the present invention, there is provided aT-cell receptor, or a T-cell displaying the T-cell receptor, which isspecific for a polypeptide as set out above, when the polypeptide ispresented on an MHC molecule.

As set out above, the polypeptide of the present invention comprises aregion of at least 12 amino acids of a self-antigen. Polypeptides ofthis length are presented on MHC class II molecules. Therefore, theT-cell receptor, or the T-cell displaying the T-cell receptor is capableof recognising and binding to a polypeptide when presented on an MHCclass II molecule. MHC class II molecules typically bind polypeptidesthat are between 12 and 24 amino acids in length. In embodiments wherethe T-cell receptor, or the T-cell displaying the T-cell receptor, isdescribed as specific for a polypeptide that is longer than 12 to 24amino acids in length, it is to be understood that an immunogenicfragment of the polypeptide is presented on the MHC molecule.

In embodiments where the self-antigen is telomerase (hTERT), it ispreferred that the T-cell receptor, or the T-cell displaying the T-cellreceptor, is specific for a polypeptide consisting of a sequenceselected from SEQ ID NOS. 1 to 5, or an immunogenic fragment thereofconsisting of at least 12 amino acids, when the polypeptide or theimmunogenic fragment thereof is presented on an MHC molecule. It isparticularly preferred that the T-cell receptor, or the T-celldisplaying the T-cell receptor, is specific for a polypeptide consistingof the sequence of SEQ ID NO. 1, 2 or 3, or an immunogenic fragmentthereof consisting of at least 12 amino acids, when the polypeptide orthe immunogenic fragment thereof is presented on an MHC molecule.

In some embodiments, there is provided a cocktail (i.e. a mixture) ofT-cell receptors, or a cocktail of T-cells displaying the T-cellreceptors. That is to say, the cocktail comprises different T-cellreceptors, or T-cells displaying the different T-cell receptors, each ofwhich is specific for a different polypeptide, when presented on an MHCmolecule.

In one embodiment, the cocktail of different T-cell receptors, or thecocktail of T-cells displaying the different T-cell receptors isspecific for different polypeptides from the same self-antigen, wheneach polypeptide is presented on an MHC molecule, or alternatively, isspecific for different polypeptides from two or more differentself-antigens, when each polypeptide is presented on an MHC molecule. Inone embodiment, the cocktail of different T-cell receptors, or thecocktail of T-cells displaying the different T-cell receptors, isspecific for at least 2 or at least 3 different polypeptides of aself-antigen, when each polypeptide is presented on an MHC molecule.That is to say, in some embodiments, the cocktail is specific for morethan 2 or more than 3 polypeptides having different sequences, when eachpolypeptide is presented on an MHC molecule (e.g. 3, 4, or 5polypeptides). It is particularly preferred that the cocktail ofdifferent T-cell receptors, or the cocktail of T-cells displaying thedifferent T-cell receptors, is specific for polypeptides capable ofbeing bound and presented by MHC class I and/or class II molecules ofmore than one HLA allele.

It is preferred that the cocktail of T-cell receptors, or the cocktailof T-cells displaying the T-cell receptors, is specific for differentpolypeptides of the hTERT protein, when each polypeptide is presented onan MHC molecule.

It is preferred that the polypeptides to which the cocktail of T-cellreceptors, or the cocktail of T-cells displaying the T-cell receptors,are specific when presented on an MHC molecule, consist of sequencesfrom at least 2 different polypeptides comprising sequences from SEQ. IDNOS. 1 to 5. It is particularly preferred that the polypeptides to whichthe cocktail of T-cell receptors, or the cocktail of T-cells displayingthe T-cell receptors, are specific when presented on an MHC molecule,consist of the sequence of SEQ. ID NOS. 1, 2 and 3. It is especiallypreferred that the polypeptides to which the cocktail of T-cellreceptors, or the cocktail of T-cells displaying the T-cell receptors,are specific when presented on an MHC molecule, consist of the sequencesof SEQ. ID NOS. 1,2 and 3.

In some embodiments, a polypeptide to which the cocktail of T-cellreceptors, or the cocktail of T-cells displaying the T-cell receptors,is specific is an immunogenic fragment of that polypeptide, and theimmunogenic fragment is presented on the MHC molecule. It is to beunderstood that certain aforementioned polypeptides, such as SEQ ID NO.1, are longer than would normally be accommodated on an MHC class IImolecule. Therefore, in embodiments in which a T-cell receptor, or aT-cell displaying the T-cell receptor, or a cocktail thereof, isdescribed as specific for a polypeptide comprising or consisting of thesequence of SEQ ID NO. 1, it is to be understood that an immunogenicfragment, comprising at least 12 amino acids of SEQ ID NO. 1, ispresented on the MHC molecule.

In alternative variants, the sequence of the polypeptide to which the oreach T-cell receptor, or the or each T-cell displaying the T-cellreceptor, is specific when bound to an MHC molecule is not identical tothat aforementioned but instead has at least 80%, 90%, 95% or 99%sequence identity thereto, provided that the polypeptide is stillcapable of being presented by the MHC molecule.

Immune Checkpoint Inhibitor

The second component of the kit for the treatment of cancer is an immunecheckpoint inhibitor.

In the present invention, an immune checkpoint inhibitor is anycompound, substance or composition (e.g. any small molecule chemicalcompound, antibody, nucleic acid molecule, or polypeptide, or fragmentsthereof) that is capable of down-regulating or blocking an immunecheckpoint to allow more extensive immune activity. It is preferred thatthe immune checkpoint inhibitor targets the CTLA-4 checkpoint and/or thePD-1 checkpoint. In additional embodiments, the immune checkpointinhibitor is targeted at another member of the CD28CTLA-4 Ig superfamilysuch as BTLA, LAG3, ICOS, PDL1 or KIR (Page et al., Annual Review ofMedicine 65:27 (2014)). In further additional embodiments, the immunecheckpoint inhibitor is targeted at a member of the TNFR superfamilysuch as CD40, OX40, CD137, GITR, CD27 or TIM-3. In a further embodiment,the immune checkpoint inhibitor targets Indoleamine 2,3-dioxygenase(IDO).

In some embodiments, targeting an immune checkpoint is accomplished withan inhibitory antibody, or antigen-binding fragment thereof or a similarmolecule. Examples of such suitable therapeutic agents are shown inTable 1 and Table 2 below. In a preferred embodiment, the immunecheckpoint inhibitor is an antibody that specifically binds to a proteininvolved in the immune checkpoint pathway thereby disrupting anddown-regulating the overall activity of the immune checkpoint. It isparticularly preferred that the immune checkpoint inhibitor is ananti-CTLA-4 antibody or an anti-PD-1 antibody. It is especiallypreferred that the anti-CTLA-4 antibody is ipilimumab or tremelimumab;and that the anti-PD-1 antibody is nivolumab or pembrolizumab.

In some embodiments, the immune checkpoint inhibitor is a small moleculeantagonist that interferes with and/or inhibits the activity of aprotein involved in the immune checkpoint pathway and therebydown-regulates the overall activity of the immune checkpoint. In apreferred embodiment, the small molecule antagonist targets the CTLA-4and/or PD-1 proteins in order to down-regulate the CTLA-4 and/or PD-1checkpoints (i.e. the small molecule antagonist is a small moleculeCTLA-4 antagonist or a small molecule PD-1 antagonist).

In a further embodiment, the immune checkpoint inhibitor is ananti-PD-L1 antibody (i.e. an antibody that specifically binds to PD-L1,which is an endogenous ligand of PD-1). It is preferred that theanti-PD-L1 antibody is BMS-936559 or MPDL3280A. In an alternativeembodiment, targeting an immune checkpoint is accomplished with anagonist for the target; examples of this class include the stimulatorytargets OX40 and GITR.

TABLE 1 Other immunotherapeutic agents in development Target NameIndication(s) B7.1 Galiximab Lymphoma B7H3 MGA271 Solid tumours LAG3IMP321 Solid tumours BMS-986016 Solid tumours CD137 BMS-663513 Solidtumours PF-05082566 Lymphoma KIR IPH2101 Myeloma, AML CCR4 KW-0761 ATL,CTCL CD27 CDX-1127 Solid tumours and Heme Ox40 MEDI-6469 Solid tumoursCD40 CP-870,893 Pancreatic Heme, Haematologic tumors; ATL, acute T-cellleukemia; CTCL, cutaneous T-cel lymphoma; AML, acute myeloid leukemia

TABLE 2 Agents targeting PD-1/PD-L1 in clinical development Agenttargeting PD-1 Agent targeting PD-L1 BMS-936558/MDX-1106 NivolumabBMS-936559/MDX-1105 (fully human IgG4 mAb) (fully human IgG4 mAb) CT-011Pidilizumab N/A (humanised IgG1 mAb) N/A MPDL3280A (IgG1 mAb, Fcmodified) AMP-514 MEDI4736 (fully human mAb) MK-3475Pembrolizumab N/A(humanised IgG4 mAb) N/A MSB0010718C AUNP 12 (peptide) N/A PD-1,programmed death 1 receptor, PD-L1, programmed cell death ligand 1;IgG4, immunoglobulin G4; mAb, monoclonal antibody; N/A, not available

In the first embodiment of the invention, one immune checkpointinhibitor is provided in the kit for the treatment of cancer. It ispreferred that the immune checkpoint inhibitor is an anti-CTLA-4antibody. It is especially preferred that the immune checkpointinhibitor is ipilimumab. In a second embodiment of the invention, atleast one immune checkpoint inhibitor is provided in the kit for thetreatment of cancer. In this second embodiment, first and secondcheckpoint inhibitors are provided, wherein the first and secondcheckpoint inhibitors target different immune checkpoints. It ispreferred that the first immune checkpoint inhibitor targets the CTLA-4checkpoint and the second immune checkpoint inhibitor targets the PD-1checkpoint.

CTLA-4 and Inhibitors of the CTLA-4 Pathway:

Cytotoxic T-lymphocyte-associated antigen (CTLA-4), also known as CD152, is a co-inhibitory molecule that functions to regulate T-cellactivation.

CTLA-4 was initially identified as a negative regulator on the surfaceof T-cells that was upregulated shortly after initiation of a de novoimmune response or stimulation of an existing response in order todampen the subsequent immune T-cell response and prevent auto-immunityor uncontrolled inflammation. Thus, the magnitude of the developingimmune response has been closely tied to CTLA-4 action. In certainembodiments, the anti-CTLA-4 antibody is Ipilimumab or Tremelimumab.

Checkpoint inhibitors function by modulating the immune system'sendogenous mechanisms of T cell regulation. Ipilimumab (YERVOY,Bristol-Meyers Squibb, New York, N.Y.) is a monoclonal antibody and isthe first such checkpoint inhibitor to be approved by the US Food andDrug Administration (FDA). It has become standard treatment formetastatic melanoma (Hodi et al., N. Engl. J. Med. 363:711-23. 2010;

Robert et al., N. Engl. J. Med. 364:2517-26. 2011). Ipilimumab binds andblocks inhibitory signaling mediated by the T cell surface co-inhibitorymolecule cytotoxic T lymphocyte antigen 4 (CTLA-4). Because themechanism of action is not specific to one tumor type, and because awealth of preclinical data supports the role of tumor immunesurveillance across multiple malignancies (Andre et al, Clin. CancerRes. 19:28-33. 2013; May et al. Clin. Cancer Res. 17:5233-38. 201 1),Ipilimumab is being investigated as a treatment for patients withprostate, lung, renal, and breast cancer, among other tumor types.Ipilimumab works by activating the immune system by targeting CTLA-4.Another CTLA-4-blocking antibody, Tremelimumab, continues to beinvestigated in clinical trials and has also demonstrated durableresponses in patients with melanoma (Kirkwood et al., Clin. Cancer Res.16: 1042-48. 2010; Rihas et al. J. Clin. Oncol. 31:616-22, 2013).

PD-1 and Inhibitors of the PD-1 Pathway:

Whereas CTLA-4 serves to regulate early T cell activation, ProgrammedDeath-1 (PD-1) signaling functions in part to regulate T cell activationin peripheral tissues. The PD-1 receptor refers to an immunoinhibitoryreceptor belonging to the CD28 family. PD-1 is expressed on a number ofcell types including T regs, activated B cells, and natural killer (NK)cells, and is expressed predominantly on previously activated T cells invivo, and binds to two ligands, PD-L1 and PD-L2. PD-1's endogenousligands, PD-L1 and PD-L2, are expressed in activated immune cells aswell as nonhaematopoietic cells, including tumor cells. PD-1 as usedherein is meant to include human PD-1 (hPD-1), variants, isoforms, andspecies homologs of hPD-1, and analogs having at least one commonepitope with hPD-1. The complete hPD-1 sequence can be found underGENBANK Accession No. U64863. Programmed Death Ligand-1 (PD-L1) is oneof two cell surface glycoprotein ligands for PD-1 (the other beingPD-L2) that results in downregulation of T cell activation and cytokinesecretion upon binding to PD-1. PD-L1 as used herein includes humanPD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, andanalogs having at least one common epitope with hPD-L1. The completehPD-L1 sequence can be found under GENBANK Accession No. Q9NZQ7. Tumorshave been demonstrated to escape immune surveillance by expressingPD-L1/L2, thereby suppressing tumor-infiltrating lymphocytes viaPD-1/PD-L1,2 interactions (Dong et al. Nat. Med. 8:793-800. 2002).Inhibition of these interactions with therapeutic antibodies has beenshown to enhance T cell response and stimulate antitumor activity(Freeman et al. J. Exp. Med. 192: 1027-34.2000).

As discussed above, in some embodiments, the anti-PD-1 antibody isNivolumab (CAS Registry Number: 946414-94-4). Alternative names forNivolumab include MDX-1 106, MDX-1 106-04, ONO-4538, BMS-936558.Nivolumab is a fully human IgG4 blocking monoclonal antibody againstPD-1 (Topaliam et al., N. Engl. J. Med. 366:2443-54. 2012). Nivolumabspecifically blocks PD-1, which can overcome immune resistance. Theligands for PD-1 have been identified as PD-L1 (B7-H1), which isexpressed on all haemopoietic cells and many nonhaemopoietic tissues,and PD-L2 (B7-DC), whose expression is restricted primarily to dendriticcells and macrophages (Dong, H. et al. 1999. Nat. Med. 5: 1365; Freeman,G. J. et al. 2000. J. Exp. Med. 192: 1027; Latehman, Y. et al. 2001.Nat. Immunol 2:261; Tseng, S. Y. et al. 2001. J. Exp. Med. 193:839).PD-L1 is overexpressed in many cancers and is often associated with poorprognosis (Okazaki T et al, Intern. Immun. 2007 19(7):813) (Thompson R Het al, Cancer Res 2006, 66(7):3381), the majority of tumor infiltratingT lymphocytes predominantly express PD-1, in contrast to T lymphocytesin normal tissues and peripheral blood T lymphocytes, indicating thatup-regulation of PD-1 on tumor-reactive T cells can contribute toimpaired antitumor immune responses (Blood 2009 1 14(8): 1537).Specifically, since tumor cells express PD-L1, an immunosuppressive PD-1ligand, inhibition of the interaction between PD-1 and PD-L1 can enhanceT-cell responses in vitro and mediate preclinical antitumor activity.

A number of clinical trials (Phase I, II and III) involving Nivolumabhave been conducted or are on-going. For example, in a phase I doseescalation trial, nivolumab was safe, and objective responses were16-31% across tumor types, with most responses being durable for >1 year(Topaliam et al., Presented at Annu. Meet. Am. Soc. Clin. Oncol.,Chicago, May 31 -Jun. 4, 2013). In another study, the safety andclinical activity of nivolumab (anti-PD-1, BMS-936558, Q Q-4538) incombination with ipilimumab in patients with advanced melanoma wasinvestigated (Woichok, J Clin Oncol 31, 2013 (suppl; abstr 9012 2013ASCO Annual Meeting).

Two anti-PD-L1 inhibitory antibodies, MPDL3280A (Genentech, South SanFrancisco, Calif.) and BMS-936559 (Bristol Meyers Squibb, New York,N.Y.), have undergone clinical investigation. Like nivolumab andMK-3475, these antibodies are thought to function principally byblocking PD-1/PD-L1 signaling. Unlike PD-1 antibodies, PD-L1 antibodiesspare potential interactions between PD-L2 and PD-1, but additionallyblock interactions between PD-L1 and CD80 (Park et al., 2010. Blood 316:1291-98). MPDL3280A has been evaluated in multiple tumor types, withsafety and preliminary efficacy identified in melanoma; renal cellcarcinoma; non-small cell lung carcinoma (NSCLC); and colorectal,gastric, and head/neck squamous cell carcinoma (Herbst et al. presentedat Annu. Meet Am. Soc. Clin. Oncol., Chicago, May 31 -Jun. 4, 2013).Similarly, BMS-936559 was shown to be safe and clinically active acrossmultiple tumor types in a phase I trial. MEDI-4736 is another PD-L1-blocking antibody currently in clinical development (NCT01693562).

In addition to CTLA-4 and PD-1/PD-L1, numerous other immunomodulatorytargets have been identified primarily, many with correspondingtherapeutic antibodies that are being investigated in clinical trials.Page et al. (Annu. Rev. Med. 2014.65) details targets of antibody immunemodulators in FIG. 1, incorporated by reference herein.

Additional Components

In some embodiments of the invention, there are provided additionalcomponents in the kit for the treatment of cancer.

In one embodiment, the kit further comprises a pharmaceuticallyacceptable adjuvant, diluent or excipient.

Exemplary adjuvants include Poly I:C (Hiltonol), CpG, liposomes,microspheres, virus-like particles (ISCOMS), Freund's incompleteadjuvant, aluminium phosphate, aluminium hydroxide, alum, bacterialtoxins (for example, cholera toxin and salmonella toxin). Furtherexemplary adjuvants include Imiquimod or glucopyranosyl Lipid A. Aparticularly preferred adjuvant is GM-CSF (granulocyte macrophage colonystimulating factor). Exemplary diluents and excipients includesterilised water, physiological saline, culture fluid and phosphatebuffer. Exemplary adjuvants for use in vaccines targeting the T cell armof the immune system, as in the present invention, are detailed inPetrovsky & Aguilar Immunol Cell Biol. 2004 82(5):488-96, which isincorporated herein by reference.

The polypeptide or nucleic acid molecule as described above is, incertain embodiments, coupled to an immunogenic carrier or incorporatedinto a virus or bacterium. Exemplary immunogenic carriers includekeyhole limpet haemocyanin, bovine serum albumin, ovalbumin, fowlimmunoglobulin and peptide fragments of immunogenic toxins. In oneembodiment, the nucleic acid molecule is coupled to or integrated in acarrier selected from the group consisting of dendritic cells, yeast,bacteria, viral vectors, oncolytic viruses, virus like particles,liposomes, micellar nanoparticles or gold nanoparticles.

The kit, in some embodiments, also comprises a further therapeuticingredient. Exemplary further therapeutic ingredients includeinterleukin-2 (IL2), interleukin-12 (IL12), a further polypeptide of aself-antigen or tumour associated antigen (that is to say, a polypeptideof a self-antigen or tumour associated antigen aside from thosediscussed above) chemotherapeutics, pain killers, anti-inflammatoryagents and other anti-cancer agents.

Further details of additional components of the kit may be found inRemington's Pharmaceutical Sciences and US Pharmacopoeia, 1984, MackPublishing Company, Easton, Pa., USA.

In certain embodiments, the aforementioned components of the kit areprovided in the form of a composition or a pharmaceutical compositionfor the treatment of cancer.

In one embodiment, the vaccine (i.e. the polypeptide or nucleic acidmolecule) and immune checkpoint inhibitor are injected locally from thesame syringe. In this embodiment, a much lower dose of the immunecheckpoint inhibitor is used compared to that used when the immunecheckpoint inhibitor is administered systemically (see Fransen et al.Clin Cancer Res. 2013 19(19):5381-9; Fransen et al. Oncoimmunology. 20132(11):e26493). That is to say, the immune checkpoint inhibitor will beused at a dosage that is at the lower end of the range of 1 microgram/kgto 10 mg/kg. The dosage of the vaccine is unchanged compared to when itis administered separately from the immune checkpoint inhibitor.

Methods of the Invention

In use, each component of the kit, the composition or the pharmaceuticalcomposition as explained above is administered to a patient in need oftreatment. In principle, any mode of administration of the components ofthe kit, the composition or the pharmaceutical composition may be used.

In embodiments in which the kit, the composition or the pharmaceuticalcomposition comprises a polypeptide, the polypeptide is endocytosed byantigen presenting cells, may be subject to antigen processing and isthen presented in complex with an MHC class II molecule on the cellsurface. Through interaction with T-cell receptors on the surface ofT-cells, a CD4+ T-cell response is elicited. It is to be appreciatedthat as a result of antigen processing, the polypeptide of the kit, thecomposition or the pharmaceutical composition may also be presented in acomplex with an MHC class I molecule on the cell surface and therebyelicit a CD8+ T cell response. In embodiments in which the kit, thecomposition or the pharmaceutical composition comprises a nucleic acidmolecule, the nucleic acid molecule is also endocytosed and is thentranscribed (if the nucleic acid molecule is DNA) and translated, andthe encoded polypeptide is synthesised through endogenous cellularpathways. Subsequently, the encoded polypeptide is processed andpresented on an MHC molecule in order to elicit the T-cell response, aspreviously described. Thus the kit, the composition or thepharmaceutical composition may be used as a vaccine in order to elicitCD4+ T-cell (as well as CD8+ T cell) immunity.

In embodiments in which the kit, the composition or the pharmaceuticalcomposition comprise a T-cell receptor, or a T-cell displaying theT-cell receptor, the T-cell or the T-cell receptor directly providesCD4+ T-cell (or CD8+ T-cell) immunity.

The components of the kit as explained above may be administeredsimultaneously, separately or sequentially to a patient in need oftreatment. That is to say, the components of the kit may be administeredat a different time, as well as in a substantially simultaneous manner.The term simultaneously as used herein refers to administration of oneor more agents at the same time. For example, in certain embodiments,the at least one polypeptide of a self-antigen and the immune checkpointinhibitor are administered simultaneously. Simultaneously includesadministration contemporaneously, that is during the same period oftime. In certain embodiments, the one or more agents are administeredsimultaneously in the same hour, or simultaneously in the same day. Insome embodiments, the term “sequentially” refers to the components ofthe kit being administered within 1, 3, 5, 7, 10, 30 or 60 days of eachother. In some embodiments, the term “sequentially” refers to thecomponents of the kit being administered within 2, 4 or 6 months of eachother.

As explained above, the second component of the kit (i.e. the immunecheckpoint inhibitor) is capable of down-regulating or blocking animmune checkpoint to allow more extensive immune activity. In someembodiments, it is preferred to administer the second component of thekit subsequent to the first component of the kit. In this way, thesecond component of the kit takes effect as a T-cell immune response isinitiated in response to vaccination with the first component of the kit(which, in some embodiments, is the at least one polypeptide or thenucleic acid molecule). It is preferred to administer the secondcomponent of the kit during the initiation phase of vaccination. In someembodiments, this is within 30, 21, 14, 10, 7, 5, 3 or 1 days from theinitial vaccination with the first component of the kit. Further detailson treatment regimes in accordance with embodiments of the presentinvention are described below.

Without wishing to be bound by theory, it is thought that theadministration of the second component of the kit subsequent to thefirst component of the kit and within the aforementioned timeframepromotes a rapid and effective expansion of T-cells specific to thefirst component of the kit from a population of naïve T-cells in theprimary lymphoid organs (i.e. a rapid and effective primary immuneresponse). This is thought to be because the second component of the kittakes effect as the T-cell response is developing and prevents dampeningof the response by the immune checkpoint. Therefore, a strong de novoimmune response is promoted, which translates into higher clinicalbenefit as described below. In addition, the administration of thesecond component of the kit subsequent to the first component of the kitand within the aforementioned timeframe is thought to contribute to thegeneration of an accelerated CD4+ T cell immune response.

Sequential or substantially simultaneous administration of eachcomponent of the kit can be effected by any appropriate route including,but not limited to, intradermal routes, oral routes, intravenous routes,sub-cutaneous routes, intramuscular routes, direct absorption throughmucous membrane tissues (e.g., nasal, mouth, vaginal, and rectal), andocular routes (e.g., intravitreal, intraocular, etc.). The components ofthe kit can be administered by the same route or by different routes. Inis particularly preferred that the components of the kit areadministered by injection. In one embodiment, the components of the kitare injected directly into a tumour in a patient. If the cancer to betreated is in the nose or mouth of a patient then in some embodiments,the components of the kit, the composition or the pharmaceuticalcomposition are administered by spray and inhalation.

A suitable dosage of the first component of the kit (which, in someembodiments, is the at least one polypeptide of the self-antigen or anucleic acid molecule encoding the at least one polypeptide) is between100 and 700 μg although dosages outside this range may occasionally berequired (e.g. from 1-1500 μg). A dosage of 300 μg is particularlypreferred. In one embodiment, the first component of the kit is a T-celland a dose of 10⁶ to 10¹¹ cells is provided. A suitable dosage of thesecond component of the kit (i.e. the immune checkpoint inhibitor) is 3mg/kg although other dosages may occasionally be required (e.g. from 1microgram/kg to 10 mg/kg).

In some embodiments, a treatment regimen is pursued which comprisesbetween two and five administrations of the second component of the kit(i.e. the immune checkpoint inhibitor) wherein each administration isseparated by between two and five weeks. In a preferred embodiment, atreatment regimen is pursued which comprises three administrations ofthe immune checkpoint inhibitor) wherein each administration isseparated by three weeks.

In some embodiments, the first component of the kit (which, in someembodiments, is the at least one polypeptide, the nucleic acid moleculeor the T-cell receptor or T-cell displaying the T cell receptor) isadministered to the patient according to the following treatmentregimen. The first component of the kit is administered: (i) prior tothe first administration of the immune checkpoint inhibitor; (ii) priorto each re-administration of the immune checkpoint inhibitor; and (iii)following completion of the immune checkpoint inhibitor treatmentregimen. It is preferred that multiple administrations of the firstcomponent of the kit are provided at stages (i), (ii) and (iii).

It is particularly preferred that one to five administrations of thefirst component of the kit are provided at stages (i) and (ii) in theseven days prior to the first administration or re-administration of thecheckpoint inhibitor respectively. It is especially preferred that oneto three administrations of the first component of the kit are provided.In some embodiments, the administration of the first component of thekit at stage (i) is provided between one to three days prior to thefirst administration of the checkpoint inhibitor. It is also preferredthat the first component of the kit is administered to the patientfollowing completion of the immune checkpoint inhibitor treatmentregimen on a monthly basis (i.e. stage (iii)). In an alternativeembodiment, the administration of the first component of the kit atstage (iii) is on a quarterly basis.

In one embodiment, the first component of the kit is administered withan additional component as explained above. It is particularly preferredthat the first component of the kit is administered with GM-CSF. Asuitable dosage of GM-CSF is between 50 and 100 μg. A dosage of 75 μg isparticularly preferred.

In some embodiments, the treatment regimen using the first and secondcomponents of the kit lasts for a total of 48 weeks from the firstadministration of the second component of the kit. In alternativeembodiments, the treatment regimen is shorter or longer than 48 weeks.

As previously stated, the at least one polypeptide is of a self-antigenand/or a universal tumour antigen, which are associated with a widerange of cancer types. Therefore, the efficacy of the present inventionis not limited to any particular type of cancer. In one embodiment, theself-antigen and/or a universal tumour antigen is hTERT and so inprinciple, the components of the kit, the composition or thepharmaceutical composition may be administered to a patient sufferingfrom any type of cancer in which the telomerase gene is activated. Suchcancers include but are not limited to breast cancer, prostate cancer,pancreatic cancer, colorectal cancer, lung cancer, bladder cancer,malignant melanoma, leukaemias, lymphomas, ovarian cancer, cervicalcancer and biliary tract carcinomas. However, as the telomerase enzymeis expressed in the vast majority of cancers, it is to be understood theefficacy of the invention is not limited to any particular type ofcancer.

That telomerase is expressed in the vast majority of cancers has beendemonstrated in studies such as Kim et al. Science. 1994 Dec. 23;266(5193):2011-5 and Bearss et al. Oncogene. 2000 Dec. 27;19(56):6632-41 (both are incorporated herein by reference).

Kim et al. 1994 has demonstrated that, in cultured cells representing 18different human tissues, 98 of 100 immortal and none of 22 mortalpopulations were positive for telomerase. The human tissues from whichthe immoral cell lines having telomerase activity were derived included:skin, connective, adipose, breast, lung, stomach, pancreas, ovary,cervix, kidney, bladder, colon, prostate, CNS, retina and blood. Thepresent invention would therefore be suitable for use against cancersderived from these tissues. Similarly, 90 of 101 biopsies representing12 human tumour types and none of 50 normal somatic tissues werepositive for telomerase. The human tumour types which exhibitedtelomerase activity included: hepatocellular carcinoma, colon cancer,squamous cell carcinoma (head and neck), Wilms tumor, breast cancer(ductal and lobular, node positive), breast cancer (axillary nodenegative), prostate cancer, prostatic intraepithelial neoplasia type 3,benign prostatic hyperplasia, neuroblastoma, brain tumors, lungsmall-cell carcinoma, rhabdomyosarcoma, leiomyosarcoma, hematologicalmalignancies (including acute lymphocytic leukaemia, chronic lymphocyticleukaemia, lymphoma (adult)),

Bearss et al. 2000 has furthermore demonstrated the presence oftelomerase activity in tumour cells taken directly from patients acrossa wide range of cancer types. These tumour types included: hematologicmalignancies (including acute myeloid leukaemia, acute lymphoidleukaemia, chronic myeloid leukaemia, chronic lymphoid leukaemia(early), chronic lymphoid leukaemia (late), myeloma, low-grade lymphoma,high-grade lymphoma); breast; prostate; lung (including non-small celland small cell); colon;

ovarian; head and neck; kidney; melanoma; neuroblastoma; glioblastoma;hepatocellular carcinoma; gastric; and bladder.

It is to be understood that, as telomerase is activated in theabove-mentioned cancer types, the present invention is suitable for useagainst any one of these types of cancer (and indeed any cancer type inwhich telomerase is activated). Furthermore, it is apparent that, as theactivation of telomerase is a common property shared between cancertypes, the present invention is not limited to any particular type ofcancer.

It is to be noted that some of the polypeptides of the present invention(e.g. the polypeptide of SEQ. ID NO. 1) are longer than would normallybe accommodated in either an MHC class I or class II molecule. Peptidesof this length have been shown to induce more robust immune responses,e.g by groups working on HPV and cervical cancer vaccination (Welters etal, 2008). Without wishing to be bound by theory, it is believed thatsuch polypeptides, following their administration to a patient, areendocytosed by cells, subjected to proteolytic degradation in theproteasome and then presented on an MHC class I or class II molecule.Thus such polypeptides may give rise to an MHC class I and/or an MHCclass II restricted T-cell response. It is to be appreciated that thisis demonstrated by FIG. 6 (see Example 6) because different CD4+ cellclones reactive with SEQ. ID NO. 1 recognise different peptide fragmentsfrom this 30-mer polypeptide as a result of proteolytic cleavage. It isalso to be appreciated that longer polypeptides remain extant within apatient for a greater period of time than shorter polypeptides andtherefore there is a longer period of time during which they may elicitan immune response. This is particularly significant as regards thosepolypeptides which have a relatively low MHC binding affinity.

It is also to be appreciated that individuals will generally havedeveloped some degree of immunological tolerance to polypeptides ofself-antigens through a process whereby T-cells reactive with suchpolypeptides are destroyed in the thymus of the individual during T-celldevelopment. Thus in some embodiments of the present invention,polypeptides of the present invention with a relatively low MHC bindingaffinity are desired. This is because polypeptides with lower MHCbinding affinity will have been exposed to maturing T-cells at a lowerrate and so it is less likely that all of the individual's T-cellsreactive with the polypeptide will have been deleted from theindividual's T-cell repertoire. Thus polypeptides having a relativelylow MHC binding affinity are, in some embodiments, able to overcomeimmunological tolerance more readily.

Synergistic Effect

The at least one polypeptide of a self-antigen or universal tumourantigen, which is at least 12 amino acids in length and the checkpointinhibitor produce a synergistic effect in the treatment of cancer. Inother embodiments, the nucleic acid molecule, the T-cell receptor, orthe T-cell displaying the T-cell receptor, according to the presentinvention and the immune checkpoint inhibitor produce a synergisticeffect in the treatment of cancer.

The synergistic effect in the treatment of cancer comprises: a reductionin the time required by the immune system of the patient to mount ameasurable immune response against the at least one polypeptide of aself-antigen or universal tumour antigen; the mounting of a strongimmune response to the at least one polypeptide (i.e. a StimulationIndex, SI≥3); and an improved clinical outcome (i.e. a partial orcomplete response (also known as partial or complete remission) orstable disease). In some embodiments, the synergistic effect in thetreatment of cancer also comprises the induction of a broad immuneresponse (i.e. the mounting of an immune response against 2, 3 or morevaccine components).

Without wishing to be bound by theory, it is believed that the abilityof the at least one polypeptide of the self-antigen to elicit a CD4+ Tcell response is of central importance to the synergistic effect.Referring to FIG. 1, the mechanism by which the polypeptide of thepresent invention is expected to elicit a CD4+ T cell response is shown.By using long polypeptides, CD4+ T cells are stimulated. These cellsplay a complex role in the tumour microenvironment and are able tointeract directly with tumour cells and a number of immune effectors,leading to tumour cell destruction. Dead tumour cells release moreantigen which in turn is taken up by antigen presenting cells,stimulating a second wave of T-cell immunity targeting other tumourantigens, a phenomenon called “epitope spreading”.

The combination of the polypeptide capable of eliciting a CD4+ T cellresponse and the immune checkpoint inhibition results in a fastoccurring immune response in a high proportion of patients as well asefficient augmentation of low/non-detectable immune responses in otherpatients. This results in a high clinical response rate (i.e. theproportion of patients with a partial or complete response (also knownas partial or complete remission) or stable disease. In particular, thepolypeptide of the self-antigen and/or universal tumour antigen providesa cancer-specific immune response to patients lacking such a response,and will also augment weak or suboptimal spontaneous immune response inthe patients thus greatly extending the number of patients that maybenefit clinically from immune checkpoint inhibition. The immunecheckpoint inhibition removes the negative influence of the checkpointon T cell proliferation and thus results in a more rapid and clinicallyefficient T cell response in a higher proportion of patients. Thisincludes turning negative responses to the polypeptide of theself-antigen and/or tumour associated antigen into a positive responseby allowing extended clonal expansion long after termination ofvaccination with the polypeptide.

It is to be appreciated that the present invention is particularlyuseful in the following clinical settings. First, in patient groups inwhich the patient has a tumour where spontaneous immune responses aregenerally absent (i.e. tumour indications where immune checkpointinhibition has previously failed to provide clinical benefit) and inpatients groups where only a small fraction of patients are responsiveto immune checkpoint inhibition (e.g. patients with malignant melanoma).Second, in patient groups where previous cancer vaccines havedemonstrated their capacity to elicit immune responses to long peptidevaccines and patients where cancer vaccines can be developed, but areunable to provide substantial clinical benefit despite their capacity toinduce immune responses after vaccination. In one embodiment, thepresent invention is used in patient groups where immune checkpointtherapy currently has marginal or no clinical benefit and the inventionelicits de novo immune responses following vaccination with the at leastone polypeptide of a self-antigen.

EXAMPLES

Hereinafter, the invention will be specifically described with referenceto the Examples. However, these Examples do not limit the technicalscope of the invention.

Materials and Methods

T Cell Response Assay (Proliferation by 3H-Thymidine Incorporation)

Peripheral blood mononuclear cells (PBMCs) were obtained prior to thestart of vaccination and at multiple time points after vaccination. ThePBMCs were isolated and frozen as previously described (Inderberg-Suso EM et al., Oncoimmunology 2012 1(5):670-686, which is incorporated hereinby reference). T cell cultures generated from pre- and post-vaccinationPBMCs, after one in vitro pre-stimulation with the vaccine peptide weresubsequently tested in a standardised T cell proliferation assay using3H-Thymidine incorporation as previously described (Inderberg-Suso E Met al., Oncoimmunology 2012 1(5):670-686). Irradiated autologous PBMCswere used as antigen presenting cells (APCs). T cells (50000) wereincubated with 50000 APCs with and without the relevant antigen (e.g.the combination of SEQ ID NOS. 1, 2 and 3 as well as the individualpolypeptides of SEQ ID NOS. 1, 2 and 3). T cell cultures were tested intriplicates. The standard error of the mean (SEM) was usually below 10%.T cell bulk responses were considered antigen-specific when thestimulatory index (SI; response with antigen divided by response withoutantigen) was equal to or above 3 (SI≥3).

ELISPOT Assay

The IFN-γ ELISPOT assays were performed essentially as previouslydescribed (Gjertsen M K et al. J Mol Med (Berl) 2003; 81:43-50).Monoclonal antibody against human IFN-γ (Mabtech) was diluted with PBSto a final concentration of 5 μg/ml. 96-well MultiScreen-HA plates(Millipore) were coated with antibody by adding 75 μl/well of the stocksolution and incubated overnight at 4° C. The following day, plates werestored at room temperature for 1 h before washing wells six times withPBS 200 μl/well to remove excess antibody. To block unspecific binding,plates were incubated for 1-2 h at 37° C. with 100 μl per well ofCellGro DC medium plus 10% human serum (HS; Baxter) Thawed and washedautologous PBMCs were enumerated and added to the pre-coated wells at5×105 cells/well. The responder T cells were harvested, washed,enumerated and transferred in CellGro DC medium (CellGenix) intriplicates to the wells containing autologous PBMCs at 1×10⁵ cells perwell. Negative controls with T cells only and PBMCs only and positivecontrols with T cells+PBMC+Staphylococcus enterotoxin C3 (SEC3; ToxinTechnologies) were included. After overnight incubation at 37° C. with5% CO2 in a humidified incubator, the plates were washed six times withPBS. Between the second and third wash, the plates were incubated for 10min at room temperature. To each well, 75 μl of a stock solution of 1μg/ml of biotinylated antibody against human IFN-γ (Mabtech) was addedand plates were incubated for 2 h at room temperature. Following sixrepeated washings, plates were incubated for 1 h with 75 μl per well ofstreptavidin-ALP (Mabtech) from a stock solution (diluted 1:1000 in PBSplus 1% HSA). To remove excess antibody, the wells were again washed sixtimes with PBS. Then, after adding 75 μl of substrate BCIP/NBT(Sigma-Aldrich) to each well, plates were incubated for 5-20 min. Whenspots appeared, water was added to stop the reaction. Spots wereenumerated using an automated analyzer, CTL IMMUNOSPOT S5 VERSA-02-9030(Cellular Technology Ltd).

Example 1 Polypeptides Having the Sequences of SEQ. ID NOS. 1 and 2 anda Combination of SEQ ID NOS. 1,2 and 3 are Capable of Eliciting a CD4+ TCell Response

Peripheral blood T cell responses in a melanoma patient who had beenvaccinated with SEQ ID NOS: 1, 2 and 3. The T cells were stimulated invitro with SEQ. ID NOS. 1, 2 or 3 as well as a combination of all threepolypeptides. T cell proliferation assays and ELISPOT assays wereperformed as per the Materials and Methods section as set out herein.The results are presented in FIGS. 2A and 2B and in Tables 3A to 3Cbelow. 719-20 refers to SEQ. ID NO: 1, 725 refers to SEQ. ID NO. 2, 728refers to SEQ. ID NO. 3, and hTERT1 mix refers to a combination of SEQ.ID NOS. 1, 2 and 3. A stimulation index (SI) was calculated for allpolypeptides tested in the T cell proliferation assay. SI≥3 wasconsidered positive.

TABLE 3A Results of T cell proliferation assay #02 ES Week −1 Week 4Week 7 Week 12 719-20 0.9 22.1 56.3 17.7 725 1.0 13.7 16.8 15.3 728 0.90.7 0.5 0.8 hTert 1 mix 0.9 25.2 60.0 20.1

TABLE 3B Results of ELISPOT assay T APC T + APC Sec 3 719-20 725 728hTert 1 mix Average spot conts 0 0 1 115 182 196 1 163 Standarddeviation 0.6 0 1 24 7 107 1 96

TABLE 3C Summary of data Immune response Immune response Sample timepoint Proliferation ELISPOT Visit 1 week −1 No Not done Visit 8 week 4Yes Not done Visit 10 week 7 Yes Not done Visit 13 week 12 Yes Yes

Referring to FIG. 2A, it is shown that SEQ ID NOS: 1, 2 and thecombination of SEQ ID NOS: 1, 2 and 3 elicited a strong immune responsein the melanoma patient at weeks 4, 7 and 12 following vaccination witha combination of SEQ ID NOS: 1, 2 and 3.

This assay is the standard assay for CD4+ T cell responses. Referring toFIG. 2B, it is shown that a positive immune response to SEQ ID NOS. 1, 2and the combination of SEQ. ID NOS. 1, 2 and 3 was detected using theELISPOT assay at week 12 following vaccination in the melanoma patient.This assay has mainly been developed for measuring CD8+ T cellresponses.

Therefore, SEQ. ID NOS. 1, 2 and the combination of SEQ. ID NOS. 1, 2and 3 were capable of eliciting a CD4+ T cell response in a melanomapatient.

Example 2 Immunogenicity of Polypeptide Fragments of a PolypeptideHaving a Sequence of SEQ ID NO. 1.

CD4+ T-cells were generated from two melanoma patients (patients P7 andP9) and a lung cancer patient (patient P5). The patients had notpreviously been administered a cancer vaccine. The CD4+ T cells werestimulated in vitro with SEQ. ID NO. 1 or fragments thereof comprising14 amino acids (as set out in Table 4 below). A T cell proliferationassay was performed as per the Materials and Methods section as set outherein. SI≥2 was considered positive. The results are presented in FIGS.3A-C.

TABLE 4 Polypeptide fragments of a polypeptide having asequence of SEQ ID No. 1 SEQ ID FRAGMENT NO. SEQUENCE NAME  1ALFSVLNYERARRPGLLGASVLGLDDIHRA 719-20  7 ALFSVLNYERARRP 719-20-1  8 LFSVLNYERARRPG 719-20-2  9   FSVLNYERARRPGL 719-20-3 10   SVLNYERARRPGLL 719-20-4 11     VLNYERARRPGLLG 719-20-5 12     LNYERARRPGLLGA 719-20-6 13       NYERARRPGLLGAS 719-20-7 14       YERARRPGLLGASV 719-20-8 15         ERARRPGLLGASVL 719-20-9 16         RARRPGLLGASVLG 719-20-10 17           ARRPGLLGASVLGL 719-20-1118            RRPGLLGASVLGLD 719-20-12 19             RPGLLGASVLGLDD719-20-13 20              PGLLGASVLGLDDI 719-20-14 21              GLLGASVLGLDDIH 719-20-15 22                LLGASVLGLDDIHR719-20-16 23                 LGASVLGLDDIHRA 719-20-17

Referring to FIG. 3A, the stimulation of T cell clones (clones 28-2 and5-2) taken from melanoma patient P7 by a peptide having a sequence ofSEQ ID No. 1 and by the peptide fragments, 719-20-13, 719-20-14,719-20-15, and 719-20-16 is shown. Each peptide elicited a strongresponse from clones 28-2 and 5-2. The SI of clone 28-2 wasexceptionally high and demonstrates that these peptides can select Tcell clones of unusually high activity from the T cell repertoire ofcancer patients. Since both clones were HLA-DQ6 restricted, theseresults further demonstrate that the repertoire of T cells recognisingthese peptides presented by a given HLA class-II molecule is complex.

Referring to FIG. 3B, the stimulation of T cell clones (clone 9 and 80)taken from melanoma patient P9 by a peptide having a sequence of SEQ IDNo. 1 and by the peptide fragments 719-20-2, 719-20-3, 719-20-4,719-20-5, 719-20-6, 719-20-7, 719-20-8, and 719-20-9 is shown.Particularly strong stimulation of the T cell clone 9 of melanoma P9 wasseen for peptide fragments 719-20, 719-20-3, 719-20-4, 719-20-5,719-20-6 and 719-20-7. Both of these T cell clones were HLA-DR8restricted, demonstrating again that T cells recognising the samepeptide presented by the same HLA class-II molecule are heterogeneous.

Referring to FIG. 3C, the stimulation of a T cell clone (clone 109;HLA-DR8 restricted) taken from a lung cancer patient P5 by a peptidehaving a sequence of SEQ ID No. 1 and by the peptide fragments 719-20-2,719-20-4, 719-20-5, and 719-20-6 is shown. Each peptide elicited astrong response from clone 109.

In conclusion, the peptide fragments of SEQ ID NO. 1 successfullystimulated CD4+ T cell clones from patient samples. Furthermore, 12/17peptide fragments tested were recognised between the five T cell clonestested.

Example 3 MHC Class II Binding Motifs of SEQ ID NO. 1

MHC class II binding motifs of SEQ ID NO. 1 and the immunogenicfragments of the sequence were calculated and are shown in Table 5.

TABLE 5 MHC class II binding motifs of SEQ ID NO. 1 andimmunogenic fragments thereof SEQ ID NO. Sequence MHC Binding Motif  1ALFSVLNYERARRPGLLGASVLGLDDIHRA Th (HLA-DR*01, 04, 07, 15) 24   SVLNYERARRPGLLG Th (HLA-DR*01, 04, 07, 15) 25   FSVLNYERARRPGLLTh (HLA-DR*01, 04, 07, 15) 26           ARRPGLLGASVLGLDTh (HLA-DR*01, 04, 07, 15) 27          RARRPGLLGASVLGLTh (HLA-DR*01, 04, 07, 15) 28     VLNYERARRPGLLGATh (HLA-DR*01, 04, 07, 15) 29             RPGLLGASVLGLDDITh (HLA-DR*01, 04, 07, 15) 30     VLNYERARRPGLLGATh (HLA-DR*01, 04, 07, 15)

As can be seen from Table 5, the polypeptide of SEQ. ID NO: 1 and itsimmunogenic fragments are able to bind to a wide range of HLA molecules(note that only those presenting Th epitopes are shown in Table 5).Therefore, this polypeptide is able to generate immune responses over avery broad patient population.

Example 4 Immunogenicity of Polypeptide Fragments of a PolypeptideHaving a Sequence of SEQ ID NO. 2.

CD4+ T-cells were generated from a melanoma patient (patient P7) and anovarian cancer patient (patient P1). The patients had not previouslybeen administered a cancer vaccine. The CD4+ T cells were stimulated invitro with SEQ. ID NO. 2 or fragments thereof comprising 12 amino acids(as set out in Table 6 below). A T cell proliferation assay wasperformed as per the Materials and Methods section as set out herein.SI≥2 was considered positive. The results are presented in FIG. 4.

TABLE 6 Polypeptide fragments of a polypeptide having asequence of SEQ ID No. 2 SEQ ID NO. SEQUENCE FRAGMENT NAME  2RTFVLRVRAQDPPPE 725 31 RTFVLRVRAQDP 725-1 32  TFVLRVRAQDPP 725-2 33  FVLRVRAQDPPP 725-3 34   VLRVRAQDPPPE 725-4

Referring to FIG. 4, the stimulation of T cells taken from melanomapatient P7 and from ovarian cancer P1 by a polypeptide having a sequenceof SEQ ID No. 2 and by the polypeptide fragments, 725-2 and 725-4 isshown.

The polypeptide fragments of SEQ ID NO. 2 successfully stimulated the Tcells from patient samples. Furthermore, 2/4 polypeptide fragmentstested were recognised between the cancer patients tested.

Example 5 A Polypeptide Having the Sequence of SEQ. ID NO. 3 andFragments Thereof are Capable of Eliciting a CD4+ T Cell Response

CD4+ T cells were generated from one patient with pancreatic cancer(patient P1) and one patient with glioblastoma (patient P5) who had notbeen administered a cancer vaccine. The CD4+ T cells were stimulated invitro with SEQ. ID NO. 3 or fragments thereof comprising 12 amino acids(as set out in Table 7 below). A T cell proliferation assay wasperformed as per the Materials and Methods section as set out herein. SI3 was considered positive. The results are presented in FIG. 5.

TABLE 7 Polypeptide fragments of a polypeptide having asequence of SEQ ID No. 3 SEQ ID NO. SEQUENCE FRAGMENT NAME  3AERLTSRVKALFSVL 728 35 AERLTSRVKALF 728-1 36  ERLTSRVKALFS 728-2 37  RLTSRVKALFSV 728-3 38    LTSRVKALFSVL 728-4

Referring to FIG. 5, it is shown that a polypeptide having a sequence ofSEQ ID NO. 3 and fragments thereof elicited a CD4+ T cell response in anon-vaccinated pancreatic cancer and a glioblastoma patient.Particularly strong stimulation of the CD4+ T cells was seen for peptidefragment 728-2 in the pancreatic cancer patient whereas all fragmentsstrongly stimulated cells from the glioblastoma patient.

In conclusion, SEQ ID NO. 3 and fragments thereof were capable ofstimulating CD4+ T cells in non-vaccinated pancreatic cancer andglioblastoma cancer patients.

Example 6 Polypeptide Fragments of a Polypeptide Having the Sequence ofSEQ. ID NO. 1 Are Capable of Eliciting a CD4+ T Cell Response

CD4+ T cell clones specific for SEQ. ID NO. 1 were generated from apatient that had been vaccinated with the combination of SEQ. ID NOS. 1,2 and 3 and were stimulated with an overlapping library of 14-merpeptides of SEQ. ID NO. 1. T cell clone proliferation was measured afterpeptide stimulation using a T cell response assays (proliferation by3H-Thymidine incorporation) as per the Materials and Methods. The dataare shown in FIG. 6.

Referring to FIG. 6, it is shown that CD4+ T cell clones specific forSEQ. ID NO. 1 recognised different 14-mer fragments of the SEQ. ID NO. 1polypeptide depending on HLA restriction. Therefore, vaccination withthe full-length SEQ. ID NO. 1 is capable of producing a broad CD4+ Tcell response because T cell clones of different HLA restriction arestimulated (e.g. HLA-DR and HLA-DQ restricted T cell clones).

In conclusion, fragments of a polypeptide having the sequence of SEQ IDNO. 1 were capable of eliciting a CD4+ T cell response in T cell clonesof different HLA restrictions.

Example 7 Clinical Response Data from Patients with Unresectable orMetastatic Malignant Melanoma Who Received a Cancer Vaccine inCombination with Ipilimumab

Combination treatment with an anti-CTLA-4 blocking agent and a cancervaccine (which comprised long peptides capable of inducing a cancerspecific T helper cell response) was investigated in a clinical trial.In the trial (EudraCT number: 2013-005582-39) the combination ofipilimumab and a cancer vaccine comprising a cocktail of SEQ ID NOS: 1,2 and 3 was investigated in patients with unresectable or metastaticmalignant melanoma.

Ipilimumab is a fully human monoclonal immunoglobulin specific for humancytotoxic T lymphocyte antigen 4 (CTLA-4, CD152), an immune modulatorymolecule which is expressed on a subset of activated T-cells. Theproposed mechanism of action for ipilimumab is the disruption of theinteraction of CTLA-4 with B7 co-stimulatory molecules (CD80 or CD86)expressed on antigen presenting cells, which results in inhibition ofthe down-modulatory function of CTLA-4.

The cancer vaccine comprising SEQ ID NOS: 1, 2 and 3 is an injectabletherapeutic cancer vaccine currently in development for treatment ofseveral cancer types. It consists of a mixture of three syntheticpeptides, 15 and 30 amino acids long, which represent fragments of thenaturally occurring protein, human telomerase reverse transcriptasesubunit (hTERT), and which are capable of inducing a cancer specific Thelper cell response.

Clinical Trial

Design

This was a phase I/IIa, open label, single arm, interventional trialexamining safety and tolerability for the ipilimumab/cancer vaccinecombination in patients with unresectable or metastatic malignantmelanoma.

Treatment Regime

Patients received ipilimumab and the cancer vaccine comprising SEQ IDNOS. 1, 2 and 3 together with Granulocyte Macrophage-Colony StimulatingFactor (GM-CSF). Ipilimumab was given every 3rd week for a total of 4doses. GM-CSF and the cancer vaccine were given 7, 5 and 3 days beforefirst dose of ipilimumab. The fourth dose of GM-CSF and the cancervaccine was given 11 days after first dose of ipilimumab and then 3 daysbefore each dose of ipilimumab and thereafter every 4th week for a totalof up to 9 doses of vaccine.

Results

Of the 14 first patients enrolled in this study, 12 were eligible andtreated. The patients had a mean age of 58.7 years (range 48-74). Therewere five women and seven men. Clinical response data with a follow uptime of 5 to 14 months from start of treatment was collected and isshown in Table 8. Referring to Table 8, six of the twelve patients had aclinical response, three of these had a partial response and three hadstable disease.

TABLE 8 Clinical response data Best tumour response * N = 12 CR PR SD PDDead Number of patients (%) 0 3 (25) 3 (25) 4 (33) 2 (17) * Based onclinical evaluation CR: complete response; PR: partial response; SD:stable disease; PD: progressive disease. Best tumour response is thebest response recorded during the observation time.

Discussion

The results described above give a disease control rate (the proportionof patients with partial or complete response or stable disease) of 50%.Hodi et al. 2010 have reported results from a phase 3 study in a similarpatient population where the disease control rate (best overallresponse) in the patient group receiving ipilimumab alone was 28.5%(median follow-up time was 27.8 months) and the disease control rate inthe patient group receiving ipilimumab and the cancer vaccine gp100 was20.1% (median follow-up time was 21 months) (Hodi et al. N Engl J Med.2010 363(8):711-23). Importantly, the partial response rate in thecurrent study was 25%. Hodi et al. 2010 reported partial response ratesof 5.5% and 9.5% for the ipilimumab plus Gp100 group and the ipilimumabalone group respectively. Gp100 is a cancer vaccine comprisingHLA-A*0201-restricted 9-mer peptides derived from the melanosomalprotein, glycoprotein 100 (Gp100).

Therefore, the disease control rate observed in the clinical trialabove, where patients with unresectable or metastatic malignant melanomareceived a cancer vaccine comprising three long peptides from hTERT incombination with ipilimumab was clearly higher than that observed in asimilar patient population when ipilimumab was administered alone or incombination with a cancer vaccine comprising a short (9-mer) peptidederived from gp100. In particular, the partial response rate of theclinical trial above was substantially higher than that reported by Hodiet al. 2010.

Example 8 Overall Survival Data from Patients with Unresectable orMetastatic Malignant Melanoma Who Received a Cancer Vaccine inCombination with Ipilimumab

Introduction

This Example provides further data from the clinical trial as set outunder Example 7.

Results

Of the 14 first patients enrolled in this study, 12 were eligible andtreated. The patients had a mean age of 58.7 years (range 48-74). Therewere five women and seven men.

The overall survival (OS) rate at 18 months and 12 months fromrandomization was 75% (9/12).

Median overall survival had not yet been reached. However, withavailable follow-up data for survival ranging from 18 to 28 months,median overall survival was at least 18 months. In general, overallsurvival is defined as the length of time from randomization in theclinical study until death from any cause.

Discussion

Hodi et al. 2010 have reported results from a phase 3 study in a similarpatient population where 1 year OS rate was 46% in the patient groupreceiving ipilimumab alone and 44% in the patient group receivingipilimumab and the cancer vaccine gp100 (Hodi et al. N Engl J Med. 2010363(8):711-23). Gp100 is a cancer vaccine comprisingHLA-A*0201-restricted 9-mer peptides derived from the melanosomalprotein, glycoprotein 100 (Gp100). Hodi reported median overall survivalof 10.1 months in the ipilimumab alone group and 10.0 months in theipilimumab plus gp100 group. The median follow-up time for survival was27.8 months and 21 months in the patient groups receiving ipilimumabalone and ipilimumab plus gp100 respectively.

Therefore, the 1 year overall survival and median overall survival inthe clinical trial above, where patients with unresectable or metastaticmalignant melanoma received a cancer vaccine comprising three longpeptides from hTERT in combination with ipilimumab were clearly higherthan those observed in a similar patient population when ipilimumab wasadministered alone or in combination with a cancer vaccine comprising ashort (9-mer) peptide derived from gp100.

Example 9 Induction of Immune Responses in Samples from Lung andProstate Cancer Patients Who Received a Cancer Vaccine Alone Comparedwith Melanoma Patients Who Received a Cancer Vaccine in Combination withIpilimumab

The therapeutic cancer vaccine comprising SEQ. ID NOS. 1, 2 and 3 hasbeen investigated in two phase 1/2A clinical trials in patients withlung cancer (EudraCT number: 2012-001852-20) and prostate cancer(EudraCT number: 2012-002411-26) respectively.

Combination treatment with the anti-CTLA-4 antibody ipilimumab and thecancer vaccine comprising SEQ. ID NOS. 1, 2 and 3 has been investigatedin a clinical trial in melanoma (EudraCT number: 2013-005582-39).

Treatment Regime

Lung and Prostate Cancer Trials:

The studies were open labeled dose-escalating phase I/IIa studies of thecancer vaccine comprising SEQ. ID NOS. 1, 2 and 3 in patients withandrogen-sensitive metastatic prostate cancer and non-small cell lungcancer (NSCLC) after completion of radiation therapy and/or chemotherapyrespectively. The cancer vaccine comprising SEQ. ID NOS. 1, 2 and 3 andGM-CSF was given at days 1, 3 and 5, then at week 2, 3, 4, 6 and 8followed by monthly vaccinations up to 6 months.

Melanoma Trial:

Patients with unresectable or metastatic malignant melanoma receivedipilimumab and the cancer vaccine comprising SEQ. ID NOS. 1, 2 and 3together with GM-CSF. Ipilimumab was given every 3rd week for a total of4 doses according to standard procedure. The cancer vaccine comprisingSEQ. ID NOS. 1, 2 and 3 and GM-CSF was given before and betweentreatments of ipilimumab and thereafter every 4th week for a total of upto 9 doses of vaccine. More specifically, the cancer vaccine comprisingSEQ. ID NOS. 1, 2 and 3 and GM-CSF were given 7, 5 and 3 days beforefirst dose of ipilimumab. The fourth dose of GM-CSF and the cancervaccine was given 11 days after first dose of ipilimumab and then 3 daysbefore each dose of ipilimumab and thereafter every 4th week for a totalof up to 9 doses of vaccine.

Immune Response Analysis

Immune responses were measured by a T cell response assay (proliferationby 3H-thymidine incorporation) using patient blood samples harvestedbefore, during and after treatment as per the Materials and Methods. Thespecific T-cell response was considered positive if the peptide responsewas at least 3 times the background (Stimulation Index, SI≥3) for atleast one of the vaccine peptides or the combination of the peptides.Any patient who developed a positive specific T-cell response againstany of the peptides of SEQ. ID NOS. 1, 2 or 3 during the study wasdefined as an immune responder.

Results

Immune response data following vaccination with 300 microgram of thecancer vaccine comprising SEQ. ID NOS. 1, 2 and 3 were available from 7patients in the prostate cancer study and 6 patients from the lungcancer study. Blood samples from 11 patients in the melanoma study (i.e.300 microgram of the cancer vaccine in combination with ipilimumab) werealso available for immune response analysis. The data are summarised inFIG. 7.

Referring to FIG. 7, the percentage of patients that developed apositive immune response against the vaccine at different time pointsfollowing vaccination is shown. Overall, 10/11 (91%) patients in themelanoma trial had a positive immune response. For the one patient thatdid not have a positive response, only one post vaccination blood sampleat 4 weeks was available. Overall, 86% of patients in the combinedprostate and lung cancer groups had a positive immune response. Thepatients that received the combined treatment of the cancer vaccine andipilimumab developed an immune response faster than the patients whoreceived the cancer vaccine alone. At four weeks, 55% of the patientswho received the combination of the cancer vaccine and ipilimumab had animmune response while it took 10 weeks before more than half (54%) ofthe patients who received the cancer vaccine alone developed an immuneresponse. Two patients in the melanoma study, two patients in theprostate cancer study and one patient in the lung cancer study had aspontaneous immune response to one of the vaccine peptides, which wereall strengthened by vaccination.

Therefore, the results of FIG. 7 demonstrate that patients who receivedthe combined treatment of the cancer vaccine and ipilimumab mountedimmune responses to the polypeptides of the vaccine faster than thosepatients who received the cancer vaccine alone. Overall, a higherproportion of the patients who received the combined treatment of thecancer vaccine and ipilimumab developed an immune response against oneof the polypeptides of the vaccine over the course of the study,compared with those patients who received the cancer vaccine alone.

Example 10 Combining a Cancer Vaccine and Ipilimumab Produces aSynergistic Effect in the Treatment of Cancer

The therapeutic cancer vaccine comprising SEQ. ID NOS. 1, 2 and 3 hasbeen investigated in two phase 1/2A clinical trials in patients withlung cancer (EudraCT number: 2012-001852-20) and prostate cancer(EudraCT number: 2012-002411-26) respectively.

Combination treatment with the anti-CTLA-4 antibody ipilimumab and thecancer vaccine comprising SEQ. ID. NOS. 1, 2 and 3 has been investigatedin a clinical trial in melanoma (EudraCT number: 2013-005582-39).

Treatment Regime

Lung and Prostate Cancer Trials:

The studies were open labeled dose-escalating phase I/IIa studies of thecancer vaccine comprising SEQ. ID NOS. 1, 2 and 3 in patients withandrogen-sensitive metastatic prostate cancer and NSCLC after completionof radiation therapy and/or chemotherapy respectively. The cancervaccine comprising SEQ. ID NOS. 1, 2 and 3 and GM-CSF was given at days1, 3 and 5, then weeks 2, 3, 4, 6, 8 and 10 followed by monthlyinjections up to 6 months.

Melanoma Trial:

Patients with unresectable or metastatic malignant melanoma receivedipilimumab and the cancer vaccine comprising SEQ. ID NOS. 1, 2 and 3together with GM-CSF. Ipilimumab was given every 3rd week for a total of4 doses according to standard procedure. The cancer vaccine comprisingSEQ. ID NOS. 1, 2 and 3 and GM-CSF was given before and betweentreatments of ipilimumab and thereafter every 4th week for a total of upto 9 doses of vaccine. More specifically, the cancer vaccine comprisingSEQ. ID NOS. 1, 2 and 3 and GM-CSF were given 7, 5 and 3 days beforefirst dose of ipilimumab. The fourth dose of GM-CSF and the cancervaccine was given 11 days after first dose of ipilimumab and then 3 daysbefore each dose of ipilimumab and thereafter every 4th week for a totalof up to 9 doses of vaccine.

Immune Response Analysis

Immune responses were measured by a T cell response assay (proliferationby 3H-thymidine incorporation) using patient blood samples harvestedbefore, during and after treatment as set out in the Materials andMethods. The specific T-cell response was considered positive if thepeptide response was at least 3 times the background (Stimulation Index,SI≥3) for at least one of the vaccine peptides or the combination of thepeptides. Any patient who developed a positive specific T-cell responseagainst any of the peptides of SEQ. ID NOS. 1, 2 or 3 during the studywas defined as an immune responder.

Results

Lung and Prostate Cancer Trials:

Combined data for the 300 microgram dose cohort from the lung andprostate cancer trials are shown in Table 9A. Only data for respondingpatients are included. For the 11 responding patients out of the 13vaccinated patients, an average of 7.6 cancer vaccine injections (range6 to 11) per patient were required to obtain a positive immune responseagainst at least one of the peptides of SEQ ID NOS. 1, 2 or 3 in thecancer vaccine. This corresponds to an average dose of 2.3 mg of thecancer vaccine (range 1.8 to 3.3 mg) per patient. The average strength(SI) of the peak immune response in this group of patients was 15.5(range 3.7-34.5).

TABLE 9A Data from patients in the lung and prostate clinical trialsProstate and lung cancer Patient No. of Amount Peptide Peak No.Injections (mg) IR L1 11 3.3 3.7 L2 7 2.1 15.5 L3 7 2.1 3.8 L4 9 2.719.4 L5 7 2.1 6.4 L6 8 2.4 5.8 L7 6 1.8 34.5 P1 6 1.8 39 P2 8 2.4 4.7 P38 2.4 31.6 P4 7 2.1 6.2 Avg 7.6 2.3 15.5 IR: immune response; L1-7: lungcancer patients; P1-4: prostate cancer patients

Melanoma Trial:

In this study the same cancer vaccine dose (300 microgram per injection)was used. The data are shown in Table 9B. Ten of the eleven patients inthis group mounted a positive immune response to the cancer vaccinefollowing vaccination. The average number of cancer vaccine injectionsrequired to obtain a positive immune response in the 10 patients was 5(range 3 to 7). This corresponds to an average dose of 1.5 mg of thecancer vaccine (range 0.9 to 2.1 mg) per patient. The average strength(SI) of the peak immune response in this group of patients was 20.2(range 3.9 to 56.3).

TABLE 9B Data from patients in the melanoma clinical trial Melanoma &IPI Patient No. of Amount Peptide Peak No. Injections (mg) IR 1 7 2.13.9 2 5 1.5 56.3 3 7 2.1 5.5 4 7 2.1 15.2 5 5 1.5 10.9 6 5 1.5 7.8 7 51.5 25.9 8 3 0.9 41.3 9 3 0.9 7.8 11 3 0.9 27.2 Avg 5.0 1.5 20.2 IR:immune response

Discussion

The data presented in Tables 9A and 9B clearly demonstrate a synergisticeffect when the cancer vaccine comprising SEQ. ID NOS. 1, 2 and 3 iscombined with the CTLA-4 blocking agent ipilimumab in the treatment ofcancer. This is both manifested by a significant reduction of the timerequired by the immune system of the patient to mount a measurableimmune response to the vaccine (summarised in Table 9C) and by thesubsequent strength of the immune response. In patients with a growingtumour mass, time is critical and an early immune response will beessential in getting control of the tumour. The time difference between5 injections (15 days) and 7.6 (8) injections (36 days) is thereforehighly relevant. Another important success parameter is the strength ofthe immune response. A strong immune response is more likely to have aclinical impact than a weak response, therefore the mean peak SI of 20.2seen in the combination trial compares favourably to the mean peak SI of15.5 observed when cancer vaccine was given alone.

TABLE 9C Summary of data from patient in the lung, prostate and melanomaclinical trials Number of Amount of cancer injections to vaccine (mg)Peak 1st positive injected to Immune immune response 1st positiveresponse Treatment Indication (SI ≥ 3) immune response (SI) Cancerprostate + 7.6 2.3 15.5 vaccine lung Cancer melanoma 5 1.5 20.2vaccine + ipi

In conclusion, the data from the analysis of the role of CTLA-4 blockadein combination with a long peptide-based vaccine (i.e. comprisingpolypeptides having the sequence of SEQ. ID NOS. 1, 2 and 3) providesfor the first time an example of a synergistic effect when CTLA-4blockade is combined with a peptide vaccine-induced T cell response incancer patients. This synergistic effect comprised a reduction in thetime taken for the patients to mount a positive immune response to apeptide of the vaccine; a stronger immune response; and an improvedclinical response (i.e. as demonstrated by Example 7). Overall, thesedata provide a strong rationale for a new type of cancervaccine-checkpoint inhibitor treatment that is expected to changefurther the clinical picture in cancer treatment.

Example 11 Induction of a Broad Immune Response in Samples from MelanomaPatients Who Received a Cancer Vaccine in Combination with Ipilimumab

The therapeutic cancer vaccine comprising SEQ. ID NOS. 1, 2 and 3 hasbeen investigated in two phase 1/2A clinical trials in patients withlung cancer (EudraCT number: 2012-001852-20) and prostate cancer(EudraCT number: 2012-002411-26) respectively.

Combination treatment with the anti-CTLA4 antibody ipilimumab and thecancer vaccine comprising SEQ. ID. NOS. 1, 2 and 3 has been investigatedin a clinical trial in melanoma (EudraCT number: 2013-005582-39).

Treatment Regime

Lung and Prostate Cancer Trials:

The studies were open labeled dose-escalating phase I/IIa studies of thecancer vaccine comprising SEQ. ID NOS. 1, 2 and 3 in patients withandrogen-sensitive metastatic prostate cancer and NSCLC after completionof radiation therapy and/or chemotherapy respectively. The cancervaccine comprising SEQ. ID NOS. 1, 2 and 3 and GM-CSF was given at days1, 3 and 5, then weeks 2, 3, 4, 6, 8 and 10 followed by monthlyinjections up to 6 months. There were three different dose groups with100, 300 and 700 microgram vaccine while the adjuvant dose was 75microgram GM-CSF.

Melanoma Trial:

Patients with unresectable or metastatic malignant melanoma receivedipilimumab and the cancer vaccine comprising SEQ. ID NOS. 1, 2 and 3together with GM-CSF. Ipilimumab was given every 3rd week for a total of4 doses according to standard procedure. The cancer vaccine comprisingSEQ. ID NOS. 1, 2 and 3 and GM-CSF was given before and betweentreatments of ipilimumab and thereafter every 4th week for a total of upto 9 doses of vaccine. The vaccine dose was 300 microgram while theadjuvant dose was 75 microgram GM-CSF.

Immune Response Analysis

Immune responses were measured by a T cell response assay (proliferationby 3H-thymidine incorporation) using patient blood samples harvestedbefore, during and after treatment as set out in the Materials andMethods. The specific T-cell response was considered positive if thepeptide response was at least 3 times the background (Stimulation Index,SI≥3). Immune responses were measured for each individual peptide ofSEQ. ID NOS. 1, 2 or 3.

Results

The fraction of patients with a positive immune response for all of theindividual peptides of SEQ. ID NOS. 1, 2 or 3 after vaccination ispresented in Table 10 below.

TABLE 10 Fraction of patients responding to all three Clinical studyvaccine peptides Lung cancer 4/18 (22%) Prostate cancer 3/21 (14%)Malignant melanoma 3/11 (27%)

Discussion

As discussed in Example 9, 91% of melanoma patients who received thecombined treatment of the cancer vaccine and ipilimumab developed animmune response against one of the polypeptides of the vaccine. Thepresent Example further demonstrates that a broad immune responsedeveloped in melanoma patients who received the combined treatment ofthe cancer vaccine and ipilimumab. This is manifested by a largerfraction of patients developing an immune response against all threevaccination peptides of SEQ. ID NOS. 1, 2 and 3 when vaccination wascombined with the CTLA4 blocking agent ipilimumab as compared to whenvaccination was given alone (i.e. in the prostate and lung cancerpatients). The data presented in Table 10 therefore further demonstratea synergistic effect when the cancer vaccine comprising SEQ. ID NOS. 1,2 and 3 is combined with the CTLA-4 blocking agent ipilimumab in thetreatment of cancer. A broad immune response is known to be associatedwith favourable clinical outcome (Kenter et al. N Engl J Med. 2009 Nov.5; 361(19):1838-47).

In conclusion, the data from Example 11 provide further evidence of asynergistic effect in the treatment of cancer, in the form of theinduction of a broad immune response, when the cancer vaccine comprisingSEQ. ID NOS. 1, 2 and 3 is combined with the CTLA-4 blocking agentipilimumab.

Overall, the data in the aforementioned Examples demonstrate thatcombining a long peptide cancer vaccine against a self-antigen with ananti-CTLA-4 antibody results in the following advantages compared withadministration of the vaccine alone: the number of patients respondingto the vaccine is increased (91% of evaluable patients); the responsesappear earlier and are stronger, requiring fewer vaccinations; and thereis a higher proportion of patients able to mount an immune responseagainst all 3 components of the vaccine (i.e. a broad immune response).This amplification of the vaccine response results in a higher clinicalbenefit when the combination is administered compared to when ipilimumabis administered alone.

SCHEDULE OF SEQUENCE LISTING

SEQ. ID NO. in Sequence Listing Sequence Notes  1ALFSVLNYERARRPGLLGASVLGLDDIHRA Corresponds to amino acid positions660-689 in the hTERT protein  2 RTFVLRVRAQDPPPECorresponds to amino acid positions 691-705 in the hTERT protein  3AERLTSRVKALFSVL Corresponds to amino acid positions651-665 in the hTERT protein  4 RLTSRVKALFSVLNYCorresponds to amino acid positions 653-667 in the hTERT protein  5EARPALLTSRLRFIPK Corresponds to the GV1001 peptide  6MPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRALVAQCLVCVPWhTERT amino acid sequenceDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLARCALFVLVAPSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARRRGGSASRSLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAEEATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRDCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSFFYVTETTFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMDYVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQVNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGTTLTALEAAANPALPSDFKTILD  7 ALFSVLNYERARRPFragment of SEQ ID NO: 1- Corresponds to amino acid positions660-673 in the hTERT protein  8 LFSVLNYERARRPG Fragment of SEQ ID NO: 1-Corresponds to amino acid positions 661-674 in the hTERT protein  9FSVLNYERARRPGL Fragment of SEQ ID NO: 1-Corresponds to amino acid positions 662-675 in the hTERT protein 10SVLNYERARRPGLL Fragment of SEQ ID NO: 1-Corresponds to amino acid positions 663-676 in the hTERT protein 11VLNYERARRPGLLG Fragment of SEQ ID NO: 1-Corresponds to amino acid positions 664-677 in the hTERT protein 12LNYERARRPGLLGA Fragment of SEQ ID NO: 1-Corresponds to amino acid positions 665-678 in the hTERT protein 13NYERARRPGLLGAS Fragment of SEQ ID NO: 1-Corresponds to amino acid positions 666-679 in the hTERT protein 14YERARRPGLLGASV Fragment of SEQ ID NO: 1-Corresponds to amino acid positions 667-680 in the hTERT protein 15ERARRPGLLGASVL Fragment of SEQ ID NO: 1-Corresponds to amino acid positions 668-681 in the hTERT protein 16RARRPGLLGASVLG Fragment of SEQ ID NO: 1-Corresponds to amino acid positions 669-682 in the hTERT protein 17ARRPGLLGASVLGL Fragment of SEQ ID NO: 1-Corresponds to amino acid positions 670-683 in the hTERT protein 18RRPGLLGASVLGLD Fragment of SEQ ID NO: 1-Corresponds to amino acid positions 671-684 in the hTERT protein 19RPGLLGASVLGLDD Fragment of SEQ ID NO: 1-Corresponds to amino acid positions 672-685 in the hTERT protein 20PGLLGASVLGLDDI Fragment of SEQ ID NO: 1-Corresponds to amino acid positions 673-686 in the hTERT protein 21GLLGASVLGLDDIH Fragment of SEQ ID NO: 1-Corresponds to amino acid positions 674-687 in the hTERT protein 22LLGASVLGLDDIHR Fragment of SEQ ID NO: 1-Corresponds to amino acid positions 675-688 in the hTERT protein 23LGASVLGLDDIHRA Fragment of SEQ ID NO: 1-Corresponds to amino acid positions 676-689 in the hTERT protein 24SVLNYERARRPGLLG Fragment of SEQ ID NO: 1-Corresponds to amino acid positions 663-677 in the hTERT protein 25FSVLNYERARRPGLL Fragment of SEQ ID NO: 1-Corresponds to amino acid positions 662-676 in the hTERT protein 26ARRPGLLGASVLGLD Fragment of SEQ ID NO: 1-Corresponds to amino acid positions 670-684 in the hTERT protein 27RARRPGLLGASVLGL Fragment of SEQ ID NO: 1-Corresponds to amino acid positions 669-683 in the hTERT protein 28VLNYERARRPGLLGA Fragment of SEQ ID NO: 1-Corresponds to amino acid positions 664-678 in the hTERT protein 29RPGLLGASVLGLDDI Fragment of SEQ ID NO: 1-Corresponds to amino acid positions 671-685 in the hTERT protein 30VLNYERARRPGLLGA Fragment of SEQ ID NO: 1-Corresponds to amino acid positions 664-678 in the hTERT protein 31RTFVLRVRAQDP Fragment of SEQ ID NO: 2-Corresponds to amino acid positions 691-702 in the hTERT protein 32TFVLRVRAQDPP Fragment of SEQ ID NO: 2-Corresponds to amino acid positions 692-703 in the hTERT protein 33FVLRVRAQDPPP Fragment of SEQ ID NO: 2-Corresponds to amino acid positions 693-704 in the hTERT protein 34VLRVRAQDPPPE Fragment of SEQ ID NO: 2-Corresponds to amino acid positions 694-705 in the hTERT protein 35AERLTSRVKALF Fragment of SEQ ID NO: 3-Corresponds to amino acid positions 651-662 in the hTERT protein 36ERLTSRVKALFS Fragment of SEQ ID NO: 3-Corresponds to amino acid positions 652-663 in the hTERT protein 37RLTSRVKALFSV Fragment of SEQ ID NO: 3-Corresponds to amino acid positions 653-664 in the hTERT protein 38LTSRVKALFSVL Fragment of SEQ ID NO: 3-Corresponds to amino acid positions 654-665 in the hTERT protein

1-20. (canceled)
 21. A method of treatment of or vaccination for cancerin a patient, comprising the steps of: i) inhibiting an immunecheckpoint; and ii) simultaneously, separately or sequentiallyadministering: a) at least one polypeptide comprising a region of atleast 12 amino acids of a self-antigen or a sequence having at least 80%identity to the region, wherein the at least one polypeptide is lessthan 100 amino acids in length; b) at least one nucleic acid moleculecomprising a nucleotide sequence encoding at least one polypeptidecomprising a region of at least 12 amino acids of a self-antigen or asequence having at least 80% identity to the region; c) a T-cellreceptor specific for a polypeptide consisting of at least 12 aminoacids of a self-antigen, or a sequence having at least 80% identity tothe polypeptide, when the polypeptide is presented on an MHC molecule;or d) a T-cell displaying a T-cell receptor as defined in c).
 22. Themethod of treatment according to claim 21, wherein the at least onepolypeptide, the nucleic acid molecule, the T-cell or T-cell receptor incombination with the inhibition of the immune checkpoint produce asynergistic effect in the treatment of or vaccination for cancer. 23.The method of treatment according to claim 21, wherein the or the atleast one polypeptide comprises a region of at least 15, 20, 25 or 30amino acids of a self-antigen or a sequence having at least 80% identityto the region.
 24. The method of treatment according to claim 21,wherein the self-antigen is a universal tumour antigen.
 25. The methodof treatment according to claim 24, wherein the universal tumour antigenis selected from the group consisting of telomerase reversetranscriptase, Top2alpha, survivin, and CYP1B1.
 26. The method oftreatment according to claim 21, wherein the self-antigen is telomerasereverse transcriptase and wherein the or the at least one polypeptidecomprises: i) a polypeptide comprising a sequence of SEQ ID NO. 1; ii)an immunogenic fragment of i) comprising at least 12 amino acids; oriii) a sequence having at least 80% sequence identity to i) or
 27. Themethod of treatment according to claim 26, wherein the or the at leastone polypeptide is a cocktail of polypeptides and wherein the cocktailof polypeptides further comprises: a polypeptide comprising: a) asequence of SEQ. ID NO. 2; b) an immunogenic fragment of a) comprisingat least 12 amino acids; or c) a sequence having at least 80% sequenceidentity to a) or b).
 28. The method of treatment according to claim 27,wherein the cocktail of polypeptides further comprises: a polypeptidecomprising: a) a sequence of SEQ. ID NO. 3; b) an immunogenic fragmentof a) comprising at least 12 amino acids; or c) a sequence having atleast 80% sequence identity to a) or b).
 29. The method of treatmentaccording to claim 21, wherein the inhibition of the immune checkpointis by administration of an immune checkpoint inhibitor selected from thegroup consisting of a CTLA-4 inhibitor, a PD-L1 inhibitor, and a PD-1inhibitor.
 30. The method of treatment according to claim 29, whereinthe CTLA-4 inhibitor is selected from the group consisting of ananti-CTLA-4 antibody and a small molecule CTLA-4 antagonist.
 31. Themethod of treatment according to claim 21, wherein the or the at leastone polypeptide is a cocktail of polypeptides and wherein the cocktailof polypeptides further comprises: a polypeptide comprising: a) asequence of SEQ. ID NO. 2; b) an immunogenic fragment of a) comprisingat least 12 amino acids; or a sequence having at least 80% sequenceidentity to a) or b);  and a polypeptide comprising: d) a sequence ofSEQ. ID NO. 3; e) an immunogenic fragment of d) comprising at least 12amino acids; or f) a sequence having at least 80% sequence identity tod) or e);  wherein the inhibition of the immune checkpoint is byadministration of an immune checkpoint inhibitor; and  wherein theimmune checkpoint inhibitor is a CTLA-4 inhibitor selected from thegroup consisting of an anti-CTLA-4 antibody and a small molecule CTLA-4antagonist.
 32. The method of treatment according to claim 29, whereinthe CTLA-4 inhibitor is selected from the group consisting of ipilimumaband tremelimumab.
 33. The method of treatment according to claim 29,wherein the PD-1 inhibitor is selected from the group consisting ofnivolumab and pembrolizumab.
 34. The method of treatment according toclaim 29, wherein the PD-L1 inhibitor is selected from the groupconsisting of MPDL3280A and BMS-936559.
 35. A composition, combinationor kit suitable for the treatment of or vaccination for cancer,comprising: i) a) at least one polypeptide comprising a region of atleast 12 amino acids of a self-antigen or a sequence having at least 80%identity to the region, wherein the at least one polypeptide is lessthan 100 amino acids in length; b) at least one nucleic acid moleculecomprising a nucleotide sequence encoding at least one polypeptidecomprising a region of at least 12 amino acids of a self-antigen or asequence having at least 80% identity to the region; c) a T-cellreceptor specific for a polypeptide consisting of at least 12 aminoacids of a self-antigen, or a sequence having at least 80% identity tothe polypeptide, when the polypeptide is presented on an MHC molecule;or d) a T-cell displaying a T-cell receptor as defined in c) and ii) animmune checkpoint inhibitor, wherein the at least one polypeptide, theat least one nucleic acid molecule, the T-cell or T-cell receptor incombination with the immune checkpoint inhibitor produce a synergisticeffect in the treatment of or vaccination for cancer.
 36. Thecomposition, combination or kit according to claim 35, wherein theself-antigen is telomerase reverse transcriptase and wherein the or theat least one polypeptide is a cocktail of polypeptides and wherein thecocktail of polypeptides comprises: a polypeptide comprising: a) apolypeptide comprising a sequence of SEQ ID NO. 1; b) an immunogenicfragment of a) comprising at least 12 amino acids; or c) a sequencehaving at least 80% sequence identity to b) or c);  and a polypeptidecomprising: d) a sequence of SEQ. ID NO. 2; e) an immunogenic fragmentof d) comprising at least 12 amino acids; or f) a sequence having atleast 80% sequence identity to e) or f).
 37. A composition, combinationor kit suitable for the treatment of or vaccination for cancer,comprising: i) at least one nucleic acid molecule, wherein the at leastone nucleic acid molecule comprises a nucleotide sequence encoding apolypeptide comprising a primary sequence of SEQ. ID NO. 1 or asecondary sequence having at least 80% sequence identity to the primarysequence or an immunogenic fragment of the primary sequence or thesecondary sequence comprising at least 12 amino acids; and ii) an immunecheckpoint inhibitor.
 38. The composition, combination or kit accordingto claim 37, wherein the at least one nucleic acid molecule is acocktail of nucleic acid molecules, and wherein the cocktail of nucleicacid molecules further comprises: a nucleic acid molecule comprising anucleotide sequence encoding a polypeptide comprising a primary sequenceof SEQ. ID NO. 2 or a secondary sequence having at least 80% sequenceidentity to the primary sequence or an immunogenic fragment of theprimary sequence or the secondary sequence comprising at least 12 aminoacids.
 39. A composition, combination or kit suitable for the treatmentof or vaccination for cancer, comprising: i) at least one T-cellreceptor, or at least one T-cell displaying the T-cell receptor, whereinthe T-cell receptor or T-cell is specific for a polypeptide consistingof: a) a sequence of SEQ. ID NO. 1; b) an immunogenic fragment of a)comprising at least 12 amino acids; or c) a sequence having at least 80%identity to a) or b,  when the polypeptide is presented on an MHCmolecule; and ii) an immune checkpoint inhibitor.
 40. The composition,combination or kit according to claim 39, wherein at least one T-cellreceptor is a cocktail of T-cell receptors or the at least one T-cell isa cocktail of T-cells and wherein the cocktail further comprises: aT-cell receptor, or a T-cell displaying the T-cell receptor, specificfor a polypeptide consisting of: a) a sequence of SEQ. ID NO. 2; b) animmunogenic fragment of a) comprising at least 12 amino acids; or c) asequence having at least 80% sequence identity to a) or b), when thepolypeptide is presented on an MHC molecule.